Compositions and methods for treating toxoplasmosis, cryptosporidiosis and other apicomplexan protozoan related diseases

ABSTRACT

The present disclosure is directed to compositions and methods for inhibiting either Toxoplasma gondii (T. gondii) calcium dependent protein kinases (TgCDPKs) or Cryptosporidium parvum (C. parvum) and Cryptosporidium hominus (C. hominus) calcium dependent protein kinases (CpCDPKs) using pyrazolopyrimidine and/or imidazo[1,5-a]pyrazine inhibitors, of the Formula (I), wherein the variables X, Y, Z, R1, and R3 are defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/544,606, filed Jul. 19, 2017, which is a U.S. national phase ofInternational Application No. PCT/US2016/014996, filed on Jan. 26, 2016,which claims priority to U.S. Provisional Application No. 62/131,539,filed Mar. 11, 2015 and U.S. Provisional Application No. 62/107,746,filed Jan. 26, 2015, all of which are incorporated by reference hereinin their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. 5 R01AI089441-02, P01 AI067921, R01 AI050506, R01 AI080625 and R01 GM086858,awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present disclosure is generally directed to compositions and methodsfor treating apicomplexan protozoan related disease, such astoxoplasmosis and cryptosporidiosis.

BACKGROUND OF THE INVENTION

The apicomplexan protozoans Cryptosporidium parvum and Toxoplasma gondiiare ubiquitous parasites that infect humans and domesticated animals.Recently C. hominus was recognized to be distinct from C. parvum, anddoes not appear to infect domesticated animals, but rather appearslimited to human infections. C. parvum and C. hominus are infectiousparasites of major health concern in humans as they are a common causeof illness transmitted by water. C. parvum and C. hominus infectionsresult in debilitating diarrhea that can be life-threatening inimmunocompromised patients.

Recent studies have implicated Cryptosporidium spp. in around 15-20% ofchildhood diarrheal disease in the developing world. Currently,nitazoxanide is the only approved therapy for cryptosporidiosis but itis expensive and has not been shown to be effective in treatingimmunocompromised hosts. T. gondii may be the most common infectiouseukaryotic parasite in humans, based on sero-surveys. Transmittedprimarily through undercooked meat or accidental ingestion of cat feces,T. gondii infection presents major health concerns in immunocompromisedhosts, where it causes toxoplasmic encephalitis, and in pregnancy, whereit can result in severe birth defects or miscarriage.

Sulfadiazine and pyrimethamine are the current therapies fortoxoplasmosis, but they can cause nephrotoxicity, rash, and additionalcomplications in pregnancy. Thus, new therapies for treating infectionscaused by both parasites are greatly needed. In T. gondii,calcium-regulated signaling is associated with a number of cellularfunctions such as secretion, gliding motility and host cell invasion.The proper control of intracellular calcium levels is important for hostcell invasion and T. gondii use several mechanisms for the uptake andrelease of calcium. Furthermore, this organism contains specializedcalcium-regulated signaling enzymes, including a unique family ofcalciumdependent protein kinases (CDPKs) which are present in plants,ciliates and green algae but not in animals. These kinases are believedto be mediators of secretion, invasion, and gliding motility. T. gondii,C. parvum, and C. hominus are highly related obligate intracellularparasites. While much less is known about the role of calcium signalingin C. parvum and C. hominus, it appears that many calcium-regulatedsignaling processes are conserved from T. gondii to C. parvum. C. parvumand C. hominus also possess CDPKs that are believed to play importantroles in calcium-regulated processes and they are virtually identical inthese two spp. Thus, inhibitors of C. parvum CDPKs would be expected toinhibit C. hominus CDPK.

SUMMARY OF THE INVENTION

We have recognized that the roles that CDPKs play in calcium signalingin T. gondii, C. parvum and C. hominus make this family of kinasesintriguing targets for the development of anti-parasitic agents.Previous studies have demonstrated that TgCDPK1 plays an important rolein T. gondii invasion of mammalian cells. Pharmacological agents thatselectively inhibit the catalytic activity of TgCDPK1 block parasiticinvasion of human fibroblast cells. Furthermore, we have demonstratedthat a unique sequence and structural variation in the ATP-binding cleftof TgCDPK1 provides an opportunity to develop highly selectiveinhibitors of this kinase (the U.S. Patent Publication 2013/0018040).Specifically, TgCDPK1 contains a glycine residue at the “gatekeeper”position which allows inhibitors to access a large hydrophobic pocketthat is adjacent to the site of ATP binding (Hydrophobic Pocket II).

The present disclosure is generally directed to compositions and methodsfor the treatment of apicomplexan-related disorders, including but notlimited to toxoplasmosis, caused by the infectious eukaryotic parasiteToxoplasma gondii (T. gondii), cryptosporidiosis, caused by theinfectious eukaryotic parasites Cryptosporidium parvum (C. parvum) andCryptosporidium hominus (C. hominus), malaria, caused by the eukaryoticparasites Plasmodium falciparum (P. falciparum), Plasmodium vivax (P.vivax), and Plasmodium berghei (P. berghei), neosporosis, caused by theeukaryotic parasite Neospora caninum, sarcocystosis, caused by theeukaryotic parasite Sarcocystis neuronae and other species,besnoitiosis, (originally named globidiosis) caused by Besnoitiabesnoiti, coccidiosis, caused by Hammondia hammondi and other Hammondiaspp., cystoisoporosis, caused by the eukaryotic parasitic speciesCystoisoporosis suis and other species, babesiosis, caused by theeukaryotic parasites Babesia microtii and other species, andtheileriosis, caused by the eukaryotic parasites Theileria equi andother species. Because all known human kinases possess larger residues(threonine, valine or a larger amino acid) at the gatekeeper position,the inventors have recognized that it is possible to gain selectivity bydeveloping inhibitors that optimize interactions with the enlargedATP-binding pocket of TgCDPK1. In one embodiment, the present disclosureis directed to compositions and methods for inhibiting apicomplexancalcium dependent protein kinases, including but not limited to T.gondii calcium dependent protein kinases (TgCDPKs), C. parvum and C.hominus calcium dependent protein kinases (CpCDPKs), or P. falciparumand P. berghei calcium dependent protein kinase 4 (PfCDPKs) usingpyrazolopyrimidine inhibitors, or in another embodiment,imidazo[1,5-a]pyrazine inhibitors, both classes of compounds designed tobe inactive against mammalian kinases.

In one aspect, the present disclosure provides compounds of the formula(I),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₂₋₆ alkyl, C₁₋₆ haloalkyl, —C₁₋₆ alkyl-R¹², C₃₋₈ cycloalkyl,    heterocyclyl, heteroaryl, or aryl, wherein    -   the alkyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl groups        are each optionally substituted with one or two R¹¹ groups;    -   each R¹¹ is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,        —S(O)₂NR₂, or —S(O)₂R;    -   and    -   R¹² is —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR,        —N(R)C(O)NR₂, aryl, heteroaryl, C₃₋₈ cycloalkyl, or        heterocyclyl, wherein R¹² is optionally substituted by one, two,        or three groups that are each independently halogen, cyano,        nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ hydoxyalkyl, —OR, —SR,        —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR,        —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;-   R³ is one of the formulas,

wherein

-   -   n is 0, 1, or 2;    -   Q is —O—, —S—, or —N(R^(Q))—, wherein R^(Q) is hydrogen or C₁₋₆        alkyl; and    -   R³³ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, (C₃₋₈        cycloalkyl)C₁₋₆ alkyl, or heterocyclyl, wherein the alkyl,        cycloalkyl, and heterocyclyl are optionally substituted with        one, two, three, or four groups that are each independently        halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,        —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl,    -   each R³² is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴,        —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂,        —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein        each R³⁴ is independently hydrogen or C₁₋₆ alkyl;        and

-   each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆    haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,    heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl,    arylalkyl, heteroaryl, and heteroarylalkyl are optionally    substituted with one, two, three, or four groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR⁰, —SR⁰, —N(R⁰)₂, —C(O)R⁰, —C(O)OR⁰, —C(O)N(R⁰)₂, —S(O)₂R⁰,    —OC(O)R⁰, —OC(O)OR⁰, —OC(O)N(R⁰)₂, —N(R)C(O)R⁰, —N(R⁰)C(O)OR⁰, or    —N(R⁰)C(O)N(R⁰)₂, wherein each R⁰ is independently hydrogen or C₁₋₆    alkyl.

In certain embodiments, the compound of the formula (I) is not:

-   1-(6-ethoxynaphthalen-2-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine;-   3-(6-isopropoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-ethoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-methoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-isopropoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-(piperidin-4-ylmethyl)-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(benzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-butoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(allyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(allyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-butoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-isobutoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-isobutoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(2,5-dimethylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-(2-methylbenzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-(2-methyl-5-(trifluoromethyl)benzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(3-chloro-4-(2,2,2-trifluoroethyl)benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(3-chloro-5-fluorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-(1-phenylethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(4-tert-butylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   1-isopropyl-3-(6-(pyridin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N,N-dimethylquinolin-2-amine;-   3-tert-butyl-1-(6-ethoxynaphthalen-2-yl)imidazo[1,5-a]pyrazin-8-amine;-   3-tert-butyl-1-(6-methoxynaphthalen-2-yl)imidazo[1,5-a]pyrazin-8-amine;-   3-(6-ethoxynaphthalen-2-yl)-1-(1-ethylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;-   3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;    or-   3-(6-ethoxynaphthalen-2-yl)-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.

In certain other embodiments, the compound of the formula (I) is not anyone of compounds disclosed in International Patent Publication WO2011/0094628 and/or the U.S. Patent Publication 2013/0018040, bothincorporated herein by reference in their entirety.

In another aspect, the present disclosure provides methods for treatingan apicomplexan protozoan related disease comprising providing to apatient in need of such treatment a therapeutically effective amount ofeither (i) a compound of the disclosure or (ii) a pharmaceuticalcomposition comprising a compound of the disclosure and apharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the present disclosure provides methods for treatingtoxoplasmosis, cryptosporidiosis, coccidiosis, and malaria comprisingproviding to a patient in need of such treatment a therapeuticallyeffective amount of either (i) a compound of the disclosure or (ii) apharmaceutical composition comprising a compound of the disclosure and apharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the present disclosure provides methods for treatingmalaria comprising providing to a patient in need of such treatment atherapeutically effective amount of either (i) a compound of thedisclosure or (ii) a pharmaceutical composition comprising a compound ofthe disclosure and a pharmaceutically acceptable excipient, carrier, ordiluent.

In one aspect, the present disclosure provides methods for treatingneosporosis, caused by the eukaryotic parasite Neospora caninum,sarcocystosis, caused by the eukaryotic parasite Sarcocystis neuronaeand other species, besnoitiosis, caused by Besnoitia besnoiti,coccidiosis, caused by Hammondia hammondi and other Hammondia spp.,cystoisoporosis, caused by the eukaryotic parasitic speciesCystoisoporosis suis and other species, babesiosis, caused by theeukaryotic parasites Babesia microtii and other species, andtheileriosis, caused by the eukaryotic parasites Theileria equi andother species comprising providing to a patient in need of suchtreatment a therapeutically effective amount of either (i) a compound ofthe disclosure or (ii) a pharmaceutical composition comprising acompound of the disclosure and a pharmaceutically acceptable excipient,carrier, or diluent. In some embodiments of this aspect, the compound is3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amineor1-(4-Amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows comparison of oral PK for compounds of Example 24 and 25 inmice. Both compounds were dosed at 10 mg/kg, PO.

FIG. 2 shows multiple dose PK studies with compounds of Example 24 and25. The dosing regimens were simulated based on the disposition datafollowing single oral doses to mice (lines) and the observed plasmaconcentrations (dots) compared to the simulation. (top left panel) 24was dosed at 20 mg/kg on day 1 and then 5 mg/kg every 24 hours for 5doses. (bottom left panel) 24 was dosed at 50 mg/kg every 48 hours for 5doses. (top right panel) 25 was dosed at 20 mg/kg on day 1 and then 5mg/kg every 24 hours for 5 doses. (bottom right panel) 25 was dosed at20 mg/kg on day 1 and then 10 mg/kg every 48 hours for 5 doses. Each dotis a mean of 3 mice with standard deviation.

FIG. 3 shows plasma concentration time curves for (A) compound ofExample 24 (left) and of Example 25 (right) following IV and PO dosingto rats; and (B) compound of Example 24 following IV and PO dosing todogs (left) and monkeys (right).

FIG. 4 shows EC₅₀ curves of inhibitors 24 (left) and 25 (right) for T.gondii over-expressing either wild type (wt) TgCDPK1 (black circles) ora drug resistant G128M TgCDPK1 mutant (gray squares). All experimentswere performed in triplicate.

FIG. 5 shows exposure to compound of Example 24 in multiple dosetoxicity studies. The concentration profiles of 24 following 30 mg/kgand 100 mg/kg daily doses were simulated based on the single dose 10mg/kg data. The mean observed concentrations (n=3) with standarddeviation are plotted as triangles for 30 mg/kg dosing and circles forthe 100 mg/kg dosing) with the simulation.

FIG. 6 shows activity of compound of Example 24 against acutetoxoplasmosis. Efficacy was evaluated by measurement of T. gondii inperitoneal fluid (top), spleen (middle), and brain (bottom) in twoexperiments. Mice were treated daily for 5 days, beginning 2 days afterIP infection with T. gondii. Mice were analyzed one day after the lastdose. Peritoneal fluid was analyzed by fluorescent microscopy and spleenand brain tissue were analyzed by quantitative real-time PCR. Groupsconsisted of 4 mice. Bars represent the mean and the standard error ofthe mean. PEG=polyethylene glycol; mpk=mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

Before the disclosed methods are described, it is to be understood thatthe aspects described herein are not limited to specific embodiments, orcompositions, and as such can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and, unless specifically definedherein, is not intended to be limiting.

Throughout this specification, unless the context requires otherwise,the word “comprise” and “include” and variations (e.g., “comprises,”“comprising,” “includes,” “including”) will be understood to imply theinclusion of a stated component, feature, element, or step or group ofcomponents, features, elements or steps but not the exclusion of anyother integer or step or group of integers or steps.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The term “pharmaceutical composition” is used in its widest sense,encompassing all pharmaceutically applicable compositions containing atleast one active substance, and optional carriers, adjuvants,constituents etc. The term “pharmaceutical composition” also encompassesa composition comprising the active substance in the form of derivativeor pro-drug, such as pharmaceutically acceptable salts and esters. Themanufacture of pharmaceutical compositions for different routes ofadministration falls within the capabilities of a person skilled inmedicinal chemistry.

In view of the present disclosure, the methods described herein can beconfigured by the person of ordinary skill in the art to meet thedesired need. In general, the disclosed methods provide improvedcompounds useful in the treatment of apicomplexan-related diseases.

In one aspect, the present disclosure provides compounds of the formula(I),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₂₋₆ alkyl, C₁₋₆ haloalkyl, —C₁₋₆ alkyl-R¹², C₃₋₈ cycloalkyl,    heterocyclyl, heteroaryl, or aryl, wherein    -   the alkyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl groups        are each optionally substituted with one or two R¹¹ groups;    -   each R¹¹ is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,        —S(O)₂NR₂, or S(O)₂R;    -   and    -   R¹² is —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR,        —N(R)C(O)NR₂, aryl, heteroaryl, C₃₋₈ cycloalkyl, or        heterocyclyl, wherein R¹² is optionally substituted by one, two,        or three groups that are each independently halogen, cyano,        nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ hydoxyalkyl, —OR, —SR,        —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR,        —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;-   R³ is one of the formulas,

wherein

-   -   n is 0, 1, or 2;    -   Q is —O—, —S—, or —N(R^(Q))—, wherein R^(Q) is hydrogen or C₁₋₆        alkyl; and    -   R³³ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, (C₃₋₈        cycloalkyl)C₁₋₆ alkyl, or heterocyclyl, wherein the alkyl,        cycloalkyl, and heterocyclyl are optionally substituted with        one, two, three, or four groups that are each independently        halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,        —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl,    -   each R³² is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴,        —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂,        —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein        each R³⁴ is independently hydrogen or C₁₋₆ alkyl;        and

-   each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆    haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,    heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl,    arylalkyl, heteroaryl, and heteroarylalkyl are optionally    substituted with one, two, three, or four groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR⁰, —SR⁰, —N(R⁰)₂, —C(O)R⁰, —C(O)OR⁰, —C(O)N(R⁰)₂, —S(O)₂R⁰,    —OC(O)R⁰, —OC(O)OR⁰, —OC(O)N(R⁰)₂, —N(R⁰)C(O)R⁰, —N(R⁰)C(O)OR⁰, or    —N(R⁰)C(O)N(R⁰)₂, wherein each R⁰ is independently hydrogen or C₁₋₆    alkyl.

The disclosure further comprises subgenera of formula (I) in which thesubstituents are selected as any and all combinations of one or more ofstructural formula (I), n, Q, R¹, R³, R³², and R³³ as defined herein,including without limitation, the following:

Structural Formula I is one of formulae (Ia)-(Ib):

R¹ is selected from one of the following groups (1a)-(1ii):

-   (1a) R¹ is C₂₋₄ alkyl, —C₁₋₆ alkyl-R¹², C₃₋₈ cycloalkyl,    heterocyclyl, heteroaryl, or aryl, wherein the cycloalkyl,    heterocyclyl, heteroaryl, and aryl groups are each optionally    substituted with one or two R¹¹ groups.-   (1b) R¹ is C₃₋₈ cycloalkyl, heterocyclyl, heteroaryl, or aryl,    wherein the cycloalkyl, heterocyclyl, heteroaryl, and aryl groups    are each optionally substituted with one or two R¹¹ groups.-   (1c) R¹ is C₃₋₈ cycloalkyl or a monocyclic heterocyclyl optionally    substituted with one R¹¹ group.-   (1d) R¹ is C₃₋₈ cycloalkyl.-   (1e) R¹ is monocyclic heterocyclyl optionally substituted with one    R¹¹ group.-   (1f) R¹ is piperidinyl or tetrahydropyranyl, each optionally    substituted with one R¹¹ group.-   (1g) R¹ is phenyl optionally substituted with one or two R¹¹ groups.-   (1h) R¹ is C₂₋₆ alkyl optionally substituted with one or two R¹¹    groups.-   (1i) R¹ is C₂₋₄ alkyl optionally substituted with one or two R¹¹    groups.-   (1j) R¹ is isopropyl or t-butyl.-   (1k) R¹ is t-butyl.-   (1l) R¹ is isopropyl.-   (1m) R¹ is C₂₋₆ alkyl or —C₁₋₆ alkyl-R¹².-   (1n) R¹ is —C₁₋₆ alkyl-R¹².-   (1o) R¹ is —C₁₋₄ alkyl-R¹².-   (1p) R¹ is CH₂—R¹².-   (1q) Any one of groups (1m)-(1p), wherein R¹² is —OR, —C(O)OR,    —C(O)NR₂, phenyl, monocyclic heteroaryl, C₃₋₈ cycloalkyl, or    monocyclic heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈    cycloalkyl, and heterocyclyl groups are each optionally substituted    by one, two, or three groups that are each independently halogen,    cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R,    —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,    —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1r) Any one of groups (1m)-(1p), R¹² is —OR.-   (1s) Any one of groups (1m)-(1p), R¹² is —OH.-   (1t) Any one of groups (1m)-(1p), wherein R¹² is phenyl, monocyclic    heteroaryl, C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, wherein the    phenyl, heteroaryl, C₃₋₈ cycloalkyl, and heterocyclyl groups are    each optionally substituted by one or two groups that are each    independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1u) Any one of groups (1m)-(1p), R¹² is phenyl or monocyclic    heterocyclyl, each optionally substituted by one, two, or three    groups that are each independently halogen, cyano, nitro, C₁₋₆    alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1v) Any one of groups (1m)-(1p), R¹² is monocyclic heterocyclyl    optionally substituted by one, two, or three groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1w) Any one of groups (1m)-(1p), wherein R¹² is monocyclic    heterocyclyl optionally substituted by one or two groups that are    each independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1x) Any one of groups (1m)-(1p), R¹² is piperidinyl or    tetrahydropyranyl, each optionally substituted by one or two groups    that are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆    haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1y) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1z) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆    alkenyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl,    arylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆ alkyl.-   (1aa) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1bb) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (1cc) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or —S(O)₂R.-   (1dd) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A), wherein each    R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (1ee) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.

-   (1ff) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl.

-   (1gg) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.

-   (1hh) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or—S(O)₂R.

-   (1ii) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A),wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.R³ is selected from one of the following groups (2a)-(2b):

-   -   (2a) R³ is group (R³-a).    -   (2b) R³ is group (R³-b).        Q is selected from one of the following groups (3a)-(3e):    -   (3a) Q is —O— or —(R^(Q))—.    -   (3b) Q is —O— or —N(H)—.    -   (3c) Q is —O—.    -   (3d) Q is —N(R^(Q))—.    -   (3e) Q is —N(H)—.        n and R³² are selected from one of the following groups        (4a)-(4x):    -   (4a) n is 0.    -   (4b) n is 0 or 1 and R³² is as defined for formula (I).    -   (4c) n is 0 or 1 and R³² is halogen, cyano, nitro, C₁₋₆ alkyl,        or C₁₋₆ haloalkyl.    -   (4d) n is 0 or 1 and R³² is halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (4e) n is 0 or 1 and each R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂,        —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴,        —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or        —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is independently hydrogen        or C₁₋₆ alkyl.    -   (4f) n is 0 or 1 and R³² is OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each        R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4g) n is 0 or 1 and R³² is —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂,        or S(O)₂R³⁴, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.    -   (4h) n is as defined for formula (I) and each R³² is        independently halogen, cyano, nitro, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (4i) n is as defined for formula (I) and each R³² is        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4j) n is as defined for formula (I) and each R³² is        independently —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴,        —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂,        —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein        each R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4k) n is as defined for formula (I) and each R³² is        independently —OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4l) n is as defined for formula (I) and each R³² is        independently —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, or —S(O)₂R³⁴,        wherein each R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4m) n is 1 or 2 and each R³² is as defined for formula (I).    -   (4n) n is 1 or 2 and each R³² is independently halogen, cyano,        nitro, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4o) n is 1 or 2 and each R³² is independently halogen, C₁₋₆        alkyl, or C₁₋₆ haloalkyl.    -   (4p) n is 1 or 2 and each R³² is independently —OR³⁴, —SR³⁴,        —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴,        —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴,        —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂ wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4q) n is 1 or 2 and each R³² is independently —OR³⁴, —SR³⁴,        —N(R³⁴)₂, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.    -   (4r) n is 1 or 2 and each R³² is independently —C(O)R³⁴,        —C(O)OR³⁴, —C(O)N(R³⁴)₂, or —S(O)₂R³⁴, wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4s) n is 1 and R³² is as defined for formula (I).    -   (4t) n is 1 and R³² is halogen, cyano, nitro, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (4u) n is 1 and R³² is halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4v) n is 1 and R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴,        —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴,        —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or        —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is independently hydrogen        or C₁₋₆ alkyl.    -   (4w) n is 1 and R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each R³⁴        is independently hydrogen or C₁₋₆ alkyl.    -   (4x) n is 1 and R³² is —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, or        —S(O)₂R³⁴, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.        R³³ is selected from one of the following groups (5a)-(5t):    -   (5a) R³³ is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, (C₃₋₈ cycloalkyl)C₁₋₆        alkyl, or heterocyclyl, wherein the alkyl, cycloalkyl, and        heterocyclyl are optionally substituted with one, two, three, or        four groups that are each independently halogen, cyano, nitro,        C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,        —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,        —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl.    -   (5b) R³³ is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, (C₃₋₈ cycloalkyl)C₁₋₆        alkyl, or heterocyclyl, wherein the alkyl, cycloalkyl, and        heterocyclyl are each substituted with one, two, three, or four        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,        —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,        —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl.    -   (5c) R³³ is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, (C₃₋₈ cycloalkyl)C₁₋₆        alkyl, or heterocyclyl, wherein the alkyl, cycloalkyl, and        heterocyclyl are optionally substituted with one or two groups        that are each independently halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (5d) R³³ is C₁₋₆ alkyl, C₃₋₈ cycloalkyl, (C₃₋₈ cycloalkyl)C₁₋₆        alkyl, or heterocyclyl, wherein the alkyl, cycloalkyl, and        heterocyclyl are each substituted with one or two groups that        are each independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5e) R³³ is C₃₋₈ cycloalkyl, (C₃₋₈ cycloalkyl)C₁₋₆ alkyl, or        heterocyclyl, wherein the, cycloalkyl, and heterocyclyl are        optionally substituted with one, two, three, or four groups that        are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5f) R³³ is C₃₋₈ cycloalkyl or (C₃₋₈ cycloalkyl)C₁₋₆ alkyl,        wherein the cycloalkyl is optionally substituted with one, two,        three, or four groups that are each independently halogen,        cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,        —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl.    -   (5g) R³³ is C₃₋₈ cycloalkyl, optionally substituted with one or        two groups that are each independently halogen, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (5h) R³³ is C₃₋₆ cycloalkyl, are each substituted with one or        two groups that are each independently halogen, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (5i) R³³ is C₃₋₈ cycloalkyl, optionally substituted with one,        two, three, or four groups that are each independently halogen,        cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,        —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl.    -   (5j) R³³ is C₁₋₆ alkyl or C₃₋₈ cycloalkyl, wherein the alkyl and        cycloalkyl are optionally substituted with one, two, three, or        four groups that are each independently halogen, cyano, nitro,        C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,        —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,        —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl.    -   (5k) R³³ is C₁₋₆ alkyl or C₃₋₈ cycloalkyl, wherein the alkyl and        cycloalkyl are optionally substituted with one or two groups        that are each independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5l) R³³ is C₁₋₆ alkyl or C₃₋₈ cycloalkyl, wherein the alkyl and        cycloalkyl are optionally substituted with one or two groups        that are each independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, or        —C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen or C₁₋₆        alkyl.    -   (5m) R³³ is C₁₋₆ alkyl or C₃₋₈ cycloalkyl, wherein the alkyl and        cycloalkyl are optionally substituted with one or two groups        that are each independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, or        —C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen or C₁₋₆        alkyl.    -   (5n) R³³ is C₁₋₆ alkyl or C₃₋₈ cycloalkyl.    -   (5o) R³³ is C₁₋₄ alkyl or C₃₋₆ cycloalkyl.    -   (5p) R³³ is C₁₋₄ alkyl, cyclopropyl, or cyclobutyl.    -   (5q) R³³ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl,        tert-butyl, cyclopropyl, or cyclobutyl.

Particular embodiments of this aspect of the invention include compoundsof any one of the formulae (I), (Ia), and (Ib), each as defined in eachof the following rows, wherein each entry is a group number as definedabove (e.g., (3c) refers to Q is —O—), a dash “-” indicates that thevariable is as defined for formula (I) or defined according to any oneof the applicable variable definitions (1a)-(5q) [e.g., when R¹ is adash, it can be either as defined for Formula (I) or any one ofdefinitions (1a)-(1ii)]; and an “x” indicates that the variable is notapplicable to the particular embodiment:

R¹ R³ R³³ n & R³² Q (1)-1 1h 2b 5a 4c 3a (1)-2 1n 2b 5a 4c 3a (1)-3 1p2b 5a 4c 3a (1)-4 1aa 2b 5a 4c 3a (1)-5 1h 2b 5e 4c 3a (1)-6 1n 2b 5e 4c3a (1)-7 1p 2b 5e 4c 3a (1)-8 1aa 2b 5e 4c 3a (1)-9 1h 2b 5i 4c 3a(1)-10 1n 2b 5i 4c 3a (1)-11 1p 2b 5i 4c 3a (1)-12 1aa 2b 5i 4c 3a(1)-13 1h 2b 5q 4c 3a (1)-14 1n 2b 5q 4c 3a (1)-15 1p 2b 5q 4c 3a (1)-161aa 2b 5q 4c 3a (1)-17 1h 2a 5a 4c 3a (1)-18 1n 2a 5a 4c 3a (1)-19 1p 2a5a 4c 3a (1)-20 1aa 2a 5a 4c 3a (1)-21 1h 2a 5e 4c 3a (1)-22 1n 2a 5e 4c3a (1)-23 1p 2a 5e 4c 3a (1)-24 1aa 2a 5e 4c 3a (1)-25 1h 2a 5i 4c 3a(1)-26 1n 2a 5i 4c 3a (1)-27 1p 2a 5i 4c 3a (1)-28 1aa 2a 5i 4c 3a(1)-29 1h 2a 5q 4c 3a (1)-30 1n 2a 5q 4c 3a (1)-31 1p 2a 5q 4c 3a (1)-321aa 2a 5q 4c 3a (1)-33 1h 2b 5a 4n 3a (1)-34 1n 2b 5a 4n 3a (1)-35 1p 2b5a 4n 3a (1)-36 1aa 2b 5a 4n 3a (1)-37 1h 2b 5e 4n 3a (1)-38 1n 2b 5e 4n3a (1)-39 1p 2b 5e 4n 3a (1)-40 1aa 2b 5e 4n 3a (1)-41 1h 2b 5i 4n 3a(1)-42 1n 2b 5i 4n 3a (1)-43 1p 2b 5i 4n 3a (1)-44 1aa 2b 5i 4n 3a(1)-45 1h 2b 5q 4n 3a (1)-46 1n 2b 5q 4n 3a (1)-47 1p 2b 5q 4n 3a (1)-481aa 2b 5q 4n 3a (1)-49 1h 2a 5a 4n 3a (1)-50 1n 2a 5a 4n 3a (1)-51 1p 2a5a 4n 3a (1)-52 1aa 2a 5a 4n 3a (1)-53 1h 2a 5e 4n 3a (1)-54 1n 2a 5e 4n3a (1)-55 1p 2a 5e 4n 3a (1)-56 1aa 2a 5e 4n 3a (1)-57 1h 2a 5i 4n 3a(1)-58 1n 2a 5i 4n 3a (1)-59 1p 2a 5i 4n 3a (1)-60 1aa 2a 5i 4n 3a(1)-61 1h 2a 5q 4n 3a (1)-62 1n 2a 5q 4n 3a (1)-63 1p 2a 5q 4n 3a (1)-641aa 2a 5q 4n 3a (1)-65 1h 2b 5a 4c 3c (1)-66 1n 2b 5a 4c 3c (1)-67 1p 2b5a 4c 3c (1)-68 1aa 2b 5a 4c 3c (1)-69 1h 2b 5e 4c 3c (1)-70 1n 2b 5e 4c3c (1)-71 1p 2b 5e 4c 3c (1)-72 1aa 2b 5e 4c 3c (1)-73 1h 2b 5i 4c 3c(1)-74 1n 2b 5i 4c 3c (1)-75 1p 2b 5i 4c 3c (1)-76 1aa 2b 5i 4c 3c(1)-77 1h 2b 5q 4c 3c (1)-78 1n 2b 5q 4c 3c (1)-79 1p 2b 5q 4c 3c (1)-801aa 2b 5q 4c 3c (1)-81 1h 2a 5a 4c 3c (1)-82 1n 2a 5a 4c 3c (1)-83 1p 2a5a 4c 3c (1)-84 1aa 2a 5a 4c 3c (1)-85 1h 2a 5e 4c 3c (1)-86 1n 2a 5e 4c3c (1)-87 1p 2a 5e 4c 3c (1)-88 1aa 2a 5e 4c 3c (1)-89 1h 2a 5i 4c 3c(1)-90 1n 2a 5i 4c 3c (1)-91 1p 2a 5i 4c 3c (1)-92 1aa 2a 5i 4c 3c(1)-93 1h 2a 5q 4c 3c (1)-94 1n 2a 5q 4c 3c (1)-95 1p 2a 5q 4c 3c (1)-961aa 2a 5q 4c 3c (1)-97 1h 2b 5a 4n 3c (1)-98 1n 2b 5a 4n 3c (1)-99 1p 2b5a 4n 3c (1)-100 1aa 2b 5a 4n 3c (1)-101 1h 2b 5e 4n 3c (1)-102 1n 2b 5e4n 3c (1)-103 1p 2b 5e 4n 3c (1)-104 1aa 2b 5e 4n 3c (1)-105 1h 2b 5i 4n3c (1)-106 1n 2b 5i 4n 3c (1)-107 1p 2b 5i 4n 3c (1)-108 1aa 2b 5i 4n 3c(1)-109 1h 2b 5q 4n 3c (1)-110 1n 2b 5q 4n 3c (1)-111 1p 2b 5q 4n 3c(1)-112 1aa 2b 5q 4n 3c (1)-113 1h 2a 5a 4n 3c (1)-114 1n 2a 5a 4n 3c(1)-115 1p 2a 5a 4n 3c (1)-116 1aa 2a 5a 4n 3c (1)-117 1h 2a 5e 4n 3c(1)-118 1n 2a 5e 4n 3c (1)-119 1p 2a 5e 4n 3c (1)-120 1aa 2a 5e 4n 3c(1)-121 1h 2a 5i 4n 3c (1)-122 1n 2a 5i 4n 3c (1)-123 1p 2a 5i 4n 3c(1)-124 1aa 2a 5i 4n 3c (1)-125 1h 2a 5q 4n 3c (1)-126 1n 2a 5q 4n 3c(1)-127 1p 2a 5q 4n 3c (1)-128 1aa 2a 5q 4n 3c (1)-129 1h 2b — — —(1)-130 1n 2b — — — (1)-131 1p 2b — — — (1)-132 1aa 2b — — — (1)-133 1h2a — — — (1)-134 1n 2a — — — (1)-135 1p 2a — — — (1)-136 1aa 2a — — —(1)-137 1h 2b — — 3c (1)-138 1n 2b — — 3c (1)-139 1p 2b — — 3c (1)-1401aa 2b — — 3c (1)-141 1h 2a — — 3c (1)-142 1n 2a — — 3c (1)-143 1p 2a —— 3c (1)-144 1aa 2a — — 3c (1)-145 1h 2b — — 3a (1)-146 1n 2b — — 3a(1)-147 1p 2b — — 3a (1)-148 1aa 2b — — 3a (1)-149 1h 2a — — 3a (1)-1501n 2a — — 3a (1)-151 1p 2a — — 3a (1)-152 1aa 2a — — 3a (1)-153 — — 5a —3a (1)-154 — — 5e — 3a (1)-155 — — 5i — 3a (1)-156 — — 5q — 3a (1)-157 —— 5a — 3c (1)-158 — — 5e — 3a (1)-159 — — 5i — 3a (1)-160 — — 5q — 3a(1)-161 — 2b 5a — 3a (1)-162 — 2b 5e — 3a (1)-163 — 2b 5i — 3a (1)-164 —2b 5q — 3a (1)-165 — 2b 5a — 3c (1)-166 — 2b 5e — 3a (1)-167 — 2b 5i —3a (1)-168 — 2b 5q — 3a (1)-169 — 2a 5a — 3a (1)-170 — 2a 5e — 3a(1)-171 — 2a 5i — 3a (1)-172 — 2a 5q — 3a (1)-173 — 2a 5a — 3c (1)-174 —2a 5e — 3a (1)-175 — 2a 5i — 3a (1)-176 — 2a 5q — 3a (1)-177 — 2a 5k —3a (1)-178 — 2b 5k — 3a (1)-179 — 2a 5k — 3c (1)-180 — 2b 5k — 3c(1)-181 — 2a 5m — 3a (1)-182 — 2b 5m — 3a (1)-183 — 2a 5m — 3c (1)-184 —2b 5m — 3c (1)-185 — 2a 5o — 3a (1)-186 — 2b 5o — 3a (1)-187 — 2a 5o —3c (1)-188 — 2b 5o — 3c (1)-189 — 2a 5q — 3a (1)-190 — 2b 5q — 3a(1)-191 — 2a 5q — 3c (1)-192 — 2b 5q — 3c (1)-193 — 2a 5k 4a 3a (1)-194— 2b 5k 4a 3a (1)-195 — 2a 5k 4a 3c (1)-196 — 2b 5k 4a 3c (1)-197 — 2a5m 4a 3a (1)-198 — 2b 5m 4a 3a (1)-199 — 2a 5m 4a 3c (1)-200 — 2b 5m 4a3c (1)-201 — 2a 5o 4a 3a (1)-202 — 2b 5o 4a 3a (1)-203 — 2a 5o 4a 3c(1)-204 — 2b 5o 4a 3c (1)-205 — 2a 5q 4a 3a (1)-206 — 2b 5q 4a 3a(1)-207 — 2a 5q 4a 3c (1)-208 — 2b 5q 4a 3c

In some embodiments, the compound of the disclosure is selected from:

-   1-(4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol;

-   1-(4-amino-5-(6-cyclopropoxynaphthalen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-methylpropan-2-ol;

-   3-(6-cyclopropoxynaphthalen-2-yl)-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine

-   3-(4-Amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol    and

-   3-(2-cyclopropoxyquinolin-6-yl)-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine.

One embodiment of the present disclosure provide a method of treating asubject in need of treatment for an apicomplexan-related diseasecomprising administering an effective amount of a compound of thedisclosure or any embodiment thereof, that inhibits the activity of anapicomplexan calcium dependent protein kinase (CDPK).

Particular embodiments of the present disclosure provide a method oftreating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of the disclosure, that inhibits theactivity of Cryptosporidium parvum and C. hominus calcium dependentprotein kinase 1 (CpCDPK1).

Other particular embodiments of the present disclosure provide a methodof treating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of the disclosure, that inhibits theactivity of T. gondii, calcium dependent protein kinase 1 (TgCDPK1).

Other particular embodiments of the present disclosure provide a methodof treating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of the disclosure, that inhibits theactivity of P. falciparum or P. berghei calcium dependent protein kinase4 (PfCDPK4 or PbCDPK4).

Optionally, the compound of any one the compounds of the disclosure, canbe administered in combination with a second agent, such as agentsspecific for use against the specific apicomplexan-related disorderbeing treated.

In one embodiment, the apicomplexan protozoan related disease istoxoplasmosis. As understood by one of ordinary skill in the art,toxoplasmosis can encompass a number of pathologies, including, but notlimited to, encephalitis, retinitis, lymphadenopathy, disseminateddisease, and hepatitis. Toxoplasmosis infects most genera ofwarm-blooded animals, including humans, but the primary host is thefelid (cat) family.

Cats are the definitive host for the Toxoplasma organism. Infection withthis protozoan parasite is fairly common, but actual disease caused bythis parasite is relatively rare in cats. Cats can become infected byToxoplasma by eating any of the three infective stages of the parasites.The most common route of infection is probably by ingestion of tissuecysts in infected prey or in other raw meat. Toxoplasma multiply in thesmall intestines and in approximately two to three weeks the oocysts areexcreted in the infected cat's feces. In another example, cats may betreated prophylactically for toxoplasmosis (e.g, a gastrointestinalinfection) provided by providing a therapeutically effective amount ofcompounds of the disclosure or to eliminate the chance that they wouldshed infectious Toxoplasmodia oocyts and infect their owners. In anotherembodiment, infected cats may be treated by providing a therapeuticallyeffective amount of a compound of the disclosure to treat toxoplasmosis.As will be understood by those of skill in the art, similar prophylacticand therapeutic methods for limiting development of or treatingtoxoplasmosis can be used in any animal that can be infected byToxoplasma sp.

Animals are infected by eating infected meat, by ingestion of feces of acat that has itself recently been infected, or by transmission frommother to fetus. While cats are often blamed for spreadingtoxoplasmosis, contact with raw meat is a more significant source ofhuman infections in many countries, and fecal contamination of hands isa greater risk factor. Infection has two stages (1) acute toxoplasmosis;and (2) latent toxoplasmosis. During acute toxoplasmosis, symptoms areoften influenza-like: swollen lymph nodes, or muscle aches and painsthat last for a month or more. Rarely, a patient with a fullyfunctioning immune system may develop eye damage from toxoplasmosis.Young children (15 years old or younger) and immunocompromised patients,such as those with HIV/AIDS, those taking certain types of chemotherapy,or those who have recently received an organ transplant, may developsevere toxoplasmosis. In an embodiment, a young child can be 14 yearsold or younger; or 13 years old or younger; or 12 years old or younger;or 11 years old or younger; or 10 years old or younger; or 9 years oldor younger; or 8 years old or younger; or 7 years old or younger; or 6years old or younger; or 5 years old or younger; or 4 years old oryounger; or 3 years old or younger; or 2 years old or younger; or 1 yearold or younger. This can cause damage to the brain (encephalitis) or theeyes (necrotizing retinochoroiditis). Infants infected via placentaltransmission may be born with either of these problems, or with nasalmalformations, although these complications are rare in newborns. Inmost immunocompetent patients, the infection enters a latent phase,during which only bradyzoites are present, forming cysts in nervous andmuscle tissue. Most infants who are infected while in the womb have nosymptoms at birth but may develop symptoms later in life. The mostcommon current therapy for toxoplasmosis is sulfadiazine/pyrimethaminecombination therapy, but therapy is often limited by allergic reactionsto the sulfa component, anemia and pancytopenia induced by blocking thefolate pathway. When sulfadiazine cannot be used, clindamycin may becombined with pyrimethamine but most experts feel it does not work aswell as sulfadiazine. Spiramycin has been used for toxoplasmosis duringpregnancy but has issues with low efficacy and is no longer available inthe United States. Thus few therapeutic alternatives are available.

In another embodiment, the apicomplexan protozoan related disease iscryptosporidiosis. Cryptosporidiosis is caused by infection with thesingle-celled parasite (not bacterium) Cryptosporidium parvum. Thisparasite is found in many mammals including lambs, calves, goat kids,piglets and humans. Research so far has shown two basic types, thebovine type which affects most species, and a second human type whichcauses disease in humans only. Outbreaks of human disease, where largenumbers of people are affected, are usually water-borne and usuallyassociated with the bovine type of cryptosporidium. Individual sporadiccases of cryptosporidiosis in humans are mostly (around 60%) associatedwith the human type of cryptosporidium.

Cryptosporidiosis affects the intestines of mammals and is typically anacute short-term infection. It is spread through the fecal-oral route,often through contaminated water; the main symptom is self-limitingdiarrhea in people with intact immune systems. In immunocompromisedindividuals, such as HIV/AIDS patients, the symptoms are particularlysevere and often fatal. Cryptosporidium is the organism most commonlyisolated in HIV positive patients presenting with diarrhea.Cryptosporidiosis is one of the most common waterborne diseases and isfound worldwide. The parasite is transmitted by environmentally hardymicrobial cysts (oocysts) that, once ingested, exist in the smallintestine and result in an infection of intestinal epithelial tissue.Infection is through contaminated material such as earth, water,uncooked or cross-contaminated food that has been in contact with thefeces of an infected individual or animal. It is especially prevalentamongst those in regular contact with bodies of fresh water includingrecreational water such as swimming pools. Other potential sourcesinclude insufficiently treated or insufficiently filtered water watersupplies, contaminated food, or exposure to feces. Symptoms appear fromtwo to ten days after infection, and last for up to two weeks or more.In immunocompetent people, the disease can be asymptomatic or causeacute diarrhea or persistent diarrhea that can last for a few weeks.There is often stomach pain or cramping and a low fever. Other symptomsmay include nausea, vomiting, malabsorption, and dehydration.Individuals who are asymptomatic (have no symptoms) are neverthelessinfective. Immunocompromised people, as well as very young or very oldpeople, can develop a more severe form of cryptosporidiosis. WhenCryptosporidium spreads beyond the intestine, as it can predominantly inpatients with AIDS, it can reach the lungs, middle ear, pancreas, andstomach. Thus, one symptom is pain in the right upper quadrant. Theparasite can infect the biliary tract, causing biliarycryptosporidiosis. This can result in cholecystitis and cholangitis.Current treatment is symptomatic, with fluid rehydration, electrolytecorrection and management of any pain. Nitazoxanide has beenFDA-approved for treatment of diarrhea caused by Cryptosporidium inpeople with healthy immune systems and is available by prescription,however it only shortens the duration of diarrhea by a couple of days.The effectiveness of nitazoxanide in immunosuppressed individuals isunclear and multiple trials have shown no benefit.

The inhibitors described herein may have use in other apicoplexaprotozoan related diseases, such as sarcocystosis caused by Sarcocystisneurona which is the most common cause of equine protozoalmyeloencephalitis (EPM) in horses in America and is transmitted by fecalcontamination by opossums, neurosporosis caused by Neurospora caninumwhich causes epidemic abortions in cattle and sheep and is transmittedby canine species fecal contamination and verticle transmissions frommother to calf or lamb, cystoisosporosis caused by Cystoisospora suisand causes epidemic diarrhea in pigs and other species in humans andanimals, besnoitiosis caused by Besnoitia besnoiti is found as a skinand systemic disease of cattle, coccidiosis caused by Eimeria spp.,cause infections and disease in poultry; which causes Babesiosis whichis caused by Babesia spp. and results in a malaria-like disease,theileriosis a tick transmitted disease caused by Theileria equi andother species and causes a wasting disease in horses and otherlivestock, and malaria in humans and animals caused by Plasmodium spp.In general, calcium dependent protein kinases homologous to Toxoplasmaand Cryptosporidium probably play important roles in the infection bythese pathogens as we have examples of the above compounds that areactive against the CDPKs of these apicoplexa protozoans and stopinfection by these protozoans in vitro, and in the case of S. neuronaand N. caninum, also evidence that therapy with these compoundssuccessfully cure mouse infections with these protozoa.

Neospora caninum is a cyst-forming apicomplexan parasite. It is closelyrelated to Toxoplasma gondii, but N. caninum exhibits distinctdifferences in transmission patterns, virulence, host specificity,immunogenetic aspects, and the pathology they induce. N. caninumrepresents one of the most important infectious causes of bovineabortion, stillbirth, and the birth of weak calves. The reservoir is thecanid, such as dogs and wild foxes and wolves, and cattle are infectedboth by accidental ingestion of canine feces or by vertical transmissionfrom mother cow to calf. In addition, N. caninum causes neosporosis, aneuromuscular disease in dogs. Neosporosis has also been detected in awide range of other species of livestock and wild animals.

Plasmodium calcium dependent protein kinase 4 (CDPK4) is essential forexflaggelation of microgametes, sexual reproduction and infection of themosquito host and is a potential drug target to block mosquitotransmission. Plasmodium transmission-blocking compounds that act viainhibition of PfCDPK4 have great promise in the armamentarium of malariacontrol. Plasmodium CDPK4 has a unique ATP binding site which rendersCDPK4 differentially sensitive to bumped kinase inhibitors (BKIs).TgCDPK1 and CpCDPK1 have ATP-binding pockets with an atypically smallgatekeeper residue, glycine. P. falciparum CDPK4 (PfCDPK4) has a serineresidue at the gatekeeper position, smaller than any gatekeeper inmammalian kinases, and an overall ATP-binding pocket that is verysimilar to TgCDPK1 and CpCDPK1. BKIs inhibit P. falciparum CDPK4(PfCDPK4) and prevents the exflagellation of malaria microgametes.Administration of BKIs to mice stops the transmission of P. berghei tomosquitoes. Finally, addition of BKIs to blood containing P. falciparumgametocytes stops exflagellation of microgametocytes and blocks theinfection of mosquitoes. BKIs are non-toxic, selective inhibitors thatblock malaria transmission to mosquitos, have favorable oralpharmacokinetic (PK) properties, and have a low likelihood of generatingresistance.

Thus, other particular embodiments of the present disclosure provide amethod for treating malaria comprising administering an effective amountof a compound of the disclosure that inhibits the activity of Plasmodiumfalciparum and P. berghei calcium dependent protein kinases. In oneembodiment, the compound can be administered in combination with asecond agent. In another embodiment, the subject has malaria, and thesecond agent is an anti-malarial therapeutic. The subject can be human.In further embodiments, the subject is a mammal other than a human, suchas a cat or livestock (e.g., pigs, sheep, goats, cattle).

Pharmaceutical Compositions

In some embodiments, the method comprises the administration of BKI in apharmaceutical composition having at least one pharmaceuticallyacceptable carrier, solvent, adjuvant or diluent.

The compounds described herein may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. The pharmaceutical compositions describedherein may be in a form suitable for oral use, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to anymethod known in the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preservative agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques. In some cases such coatings may be prepared by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatincapsules, wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin or oliveoil.

Formulations for oral use may also be presented as lozenges.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide palatable oral preparations. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations may also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compositions disclosed herein may be administered parenterally in asterile medium. The drug, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. Other adjuvants and modes ofadministration are well and widely known in the pharmaceutical art.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient. The daily dose can be administered in one tofour doses per day. In the case of skin conditions, it may be preferableto apply a topical preparation of compounds of this invention to theaffected area two to four times a day.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy. The compounds of thepresent invention may be administered alone or in combination with atleast one additional therapeutic agent. The compounds of the presentinvention may be combined with one or more additional therapeutic agentssimultaneously or sequentially.

Definitions

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication.

Terms used herein may be preceded and/or followed by a single dash, “-”,or a double dash, “=”, to indicate the bond order of the bond betweenthe named substituent and its parent moiety; a single dash indicates asingle bond and a double dash indicates a double bond. In the absence ofa single or double dash it is understood that a single bond is formedbetween the substituent and its parent moiety; further, substituents areintended to be read “left to right” unless a dash indicates otherwise.For example, C₁-C₆alkoxycarbonyloxy and —OC(O)C₁-C₆alkyl indicate thesame functionality; similarly arylalkyl and -alkylaryl indicate the samefunctionality.

The term “alkenyl” as used herein, means a straight or branched chainhydrocarbon containing from 2 to 10 carbons, unless otherwise specified,and containing at least one carbon-carbon double bond. Representativeexamples of alkenyl include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and3,7-dimethylocta-2,6-dienyl.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms, unless otherwisespecified. Representative examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl. When an “alkyl” group is a linking group between two othermoieties, then it may also be a straight or branched chain; examplesinclude, but are not limited to —CH₂—, —CH₂CH₂—, —CH₂CH₂CHC(CH₃)—,—CH₂CH(CH₂CH₃)CH₂—.

The term “alkynyl” as used herein, means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “aryl,” as used herein, means a phenyl (i.e., monocyclic aryl),a bicyclic ring system containing at least one phenyl ring or anaromatic bicyclic ring containing only carbon atoms in the aromaticbicyclic ring system or a multicyclic aryl ring system, provided thatthe bicyclic or multicyclic aryl ring system does not contain aheteroaryl ring when fully aromatic. The bicyclic aryl can be azulenyl,naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocycliccycloalkenyl, or a monocyclic heterocyclyl. The bicyclic aryl isattached to the parent molecular moiety through any carbon atomcontained within the phenyl portion of the bicyclic system, or anycarbon atom with the napthyl or azulenyl ring. The fused monocycliccycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl areoptionally substituted with one or two oxo and/or thia groups.Representative examples of the bicyclic aryls include, but are notlimited to, azulenyl, naphthyl, dihydroinden-1-yl, dihydroinden-2-yl,dihydroinden-3-yl, dihydroinden-4-yl, 2,3-dihydroindol-4-yl,2,3-dihydroindol-5-yl, 2,3-dihydroindol-6-yl, 2,3-dihydroindol-7-yl,inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl,dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl,5,6,7,8-tetrahydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl,2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl,2,3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl,benzo[d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl,2H-chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H-chromen-2-on-7-yl,2H-chromen-2-on-8-yl, isoindoline-1,3-dion-4-yl,isoindoline-1,3-dion-5-yl, inden-1-on-4-yl, inden-1-on-5-yl,inden-1-on-6-yl, inden-1-on-7-yl, 2,3-dihydrobenzo[b][1,4]dioxan-5-yl,2,3-dihydrobenzo[b][1,4]dioxan-6-yl,2H-benzo[b][1,4]oxazin3(4H)-on-5-yl,2H-benzo[b][1,4]oxazin3(4H)-on-6-yl,2H-benzo[b][1,4]oxazin3(4H)-on-7-yl,2H-benzo[b][1,4]oxazin3(4H)-on-8-yl, benzo[d]oxazin-2(3H)-on-5-yl,benzo[d]oxazin-2(3H)-on-6-yl, benzo[d]oxazin-2(3H)-on-7-yl,benzo[d]oxazin-2(3H)-on-8-yl, quinazolin-4(3H)-on-5-yl,quinazolin-4(3H)-on-6-yl, quinazolin-4(3H)-on-7-yl,quinazolin-4(3H)-on-8-yl, quinoxalin-2(1H)-on-5-yl,quinoxalin-2(1H)-on-6-yl, quinoxalin-2(1H)-on-7-yl,quinoxalin-2(1H)-on-8-yl, benzo[d]thiazol-2(3H)-on-4-yl,benzo[d]thiazol-2(3H)-on-5-yl, benzo[d]thiazol-2(3H)-on-6-yl, andbenzo[d]thiazol-2(3H)-on-7-yl. In certain embodiments, the bicyclic arylis (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 memberedmonocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl,cycloalkenyl, and heterocyclyl groups are optionally substituted withone or two groups which are independently oxo or thia. Multicyclic arylgroups are a phenyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other ring systems independently selected from the groupconsisting of a phenyl, a bicyclic aryl, a monocyclic or bicycliccycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic orbicyclic heterocyclyl, provided that when the base ring is fused to abicyclic cycloalkyl, bicyclic cycloalkenyl, or bicyclic heterocyclyl,then the base ring is fused to the base ring of the a bicycliccycloalkyl, bicyclic cycloalkenyl, or bicyclic heterocyclyl. Themulticyclic aryl is attached to the parent molecular moiety through anycarbon atom contained within the base ring. In certain embodiments,multicyclic aryl groups are a phenyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two other ring systems independently selected fromthe group consisting of a phenyl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl, provided that when the basering is fused to a bicyclic cycloalkyl, bicyclic cycloalkenyl, orbicyclic heterocyclyl, then the base ring is fused to the base ring ofthe a bicyclic cycloalkyl, bicyclic cycloalkenyl, or bicyclicheterocyclyl. Examples of multicyclic aryl groups include but are notlimited to anthracen-9-yl and phenanthren-9-yl.

The term “arylalkyl” and “-alkylaryl” as used herein, means an arylgroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofarylalkyl include, but are not limited to, benzyl, 2-phenylethyl,3-phenylpropyl, and 2-naphth-2-ylethyl.

The terms “cyano” and “nitrile” as used herein, mean a —CN group.

The term “cycloalkyl” as used herein, means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. Monocyclic ring systems are cyclichydrocarbon groups containing from 3 to 8 carbon atoms, where suchgroups can be saturated or unsaturated, but not aromatic. In certainembodiments, cycloalkyl groups are fully saturated. Examples ofmonocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fusedbicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkylring where two non-adjacent carbon atoms of the monocyclic ring arelinked by an alkylene bridge of between one and three additional carbonatoms (i.e., a bridging group of the form —(CH₂)_(w)—, where w is 1, 2,or 3). Representative examples of bicyclic ring systems include, but arenot limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, andbicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain amonocyclic cycloalkyl ring fused to either a phenyl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or amonocyclic heteroaryl. The bridged or fused bicyclic cycloalkyl isattached to the parent molecular moiety through any carbon atomcontained within the monocyclic cycloalkyl ring. Cycloalkyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In certain embodiments, the fused bicyclic cycloalkyl is a5 or 6 membered monocyclic cycloalkyl ring fused to either a phenylring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 memberedmonocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a5 or 6 membered monocyclic heteroaryl, wherein the fused bicycliccycloalkyl is optionally substituted by one or two groups which areindependently oxo or thia. Multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other rings systems independentlyselected from the group consisting of a phenyl, a bicyclic aryl, amonocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl,a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. The multicyclic cycloalkyl is attached to the parentmolecular moiety through any carbon atom contained within the base ring.In certain embodiments, multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other rings systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl,a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl. Examples of multicyclic cycloalkyl groups include, but arenot limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl,and perhydrophenoxazin-1-yl.

“Cycloalkenyl” as used herein refers to a monocyclic, bicyclic, or amulticyclic cycloalkenyl ring system. Monocyclic ring systems are cyclichydrocarbon groups containing from 3 to 8 carbon atoms, where suchgroups are unsaturated (i.e., containing at least one annularcarbon-carbon double bond), but not aromatic. Examples of monocyclicring systems include cyclopentenyl and cyclohexenyl. Bicycliccycloalkenyl rings are bridged monocyclic rings or fused bicyclic rings.Bridged monocyclic rings contain a monocyclic cycloalkenyl ring wheretwo non-adjacent carbon atoms of the monocyclic ring are linked by analkylene bridge of between one and three additional carbon atoms (i.e.,a bridging group of the form —(CH₂)_(w)—, where w is 1, 2, or 3).Representative examples of bicyclic cycloalkenyls include, but are notlimited to, norbornenyl and bicyclo[2.2.2]oct-2-enyl. Fused bicycliccycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fusedto either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl,a monocyclic heterocyclyl, or a monocyclic heteroaryl. The bridged orfused bicyclic cycloalkenyl is attached to the parent molecular moietythrough any carbon atom contained within the monocyclic cycloalkenylring. Cycloalkenyl groups are optionally substituted with one or twogroups which are independently oxo or thia. Multicyclic cycloalkenylrings contain a monocyclic cycloalkenyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two rings systemsindependently selected from the group consisting of a phenyl, a bicyclicaryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicycliccycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic orbicyclic heterocyclyl. The multicyclic cycloalkenyl is attached to theparent molecular moiety through any carbon atom contained within thebase ring. In certain embodiments, multicyclic cycloalkenyl ringscontain a monocyclic cycloalkenyl ring (base ring) fused to either (i)one ring system selected from the group consisting of a bicyclic aryl, abicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, anda bicyclic heterocyclyl; or (ii) two rings systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl,a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl.

The term “halo” or “halogen” as used herein, means —Cl, —Br, —I or —F.

The term “haloalkyl” as used herein, means at least one halogen, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of haloalkyl include,but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl,pentafluoroethyl, and 2-chloro-3-fluoropentyl.

The term “heteroaryl,” as used herein, means a monocyclic, bicyclic, ora multicyclic heteroaryl ring system. The monocyclic heteroaryl can be a5 or 6 membered ring. The 5 membered ring consists of two double bondsand one, two, three or four nitrogen atoms and optionally one oxygen orsulfur atom. The 6 membered ring consists of three double bonds and one,two, three or four nitrogen atoms. The 5 or 6 membered heteroaryl isconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the heteroaryl. Representative examplesof monocyclic heteroaryl include, but are not limited to, furyl,imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclicheteroaryl consists of a monocyclic heteroaryl fused to a phenyl, amonocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl orheterocyclyl portion of the bicyclic heteroaryl group is optionallysubstituted with one or two groups which are independently oxo or thia.When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl,or heterocyclyl ring, then the bicyclic heteroaryl group is connected tothe parent molecular moiety through any carbon or nitrogen atomcontained within the monocyclic heteroaryl portion of the bicyclic ringsystem. When the bicyclic heteroaryl is a monocyclic heteroaryl fused toa phenyl ring or a monocyclic heteroaryl, then the bicyclic heteroarylgroup is connected to the parent molecular moiety through any carbonatom or nitrogen atom within the bicyclic ring system. Representativeexamples of bicyclic heteroaryl include, but are not limited to,benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl,benzoxathiadiazolyl, benzothiazolyl, cinnolinyl,5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl,indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl,5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl,5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl,thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl, and6,7-dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain embodiments,the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroarylring fused to either a phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. The multicyclic heteroaryl group is a monocyclic heteroarylring (base ring) fused to either (i) one ring system selected from thegroup consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclicheterocyclyl, a bicyclic cycloalkenyl, and a bicyclic cycloalkyl; or(ii) two ring systems selected from the group consisting of a phenyl, abicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic orbicyclic heterocyclyl, a monocyclic or bicyclic cycloalkenyl, and amonocyclic or bicyclic cycloalkyl. The multicyclic heteroaryl group isconnected to the parent molecular moiety through any carbon atom ornitrogen atom contained within the base ring. In certain embodiments,multicyclic heteroaryl groups are a monocyclic heteroaryl ring (basering) fused to either (i) one ring system selected from the groupconsisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclicheterocyclyl, a bicyclic cycloalkenyl, and a bicyclic cycloalkyl; or(ii) two ring systems selected from the group consisting of a phenyl, amonocyclic heteroaryl, a monocyclic heterocyclyl, a monocycliccycloalkenyl, and a monocyclic cycloalkyl. Examples of multicyclicheteroaryls include, but are not limited to5H-[1,2,4]triazino[5,6-b]indol-5-yl,2,3,4,9-tetrahydro-1H-carbazol-9-yl, 9H-pyrido[3,4-b]indol-9-yl,9H-carbazol-9-yl, acridin-9-yl,

The term “heteroarylalkyl” and “-alkylheteroaryl” as used herein, meansa heteroaryl, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, fur-3-ylmethyl,1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl,pyridin-3-ylmethyl, pyridin-4-ylmethyl, pyrimidin-5-ylmethyl,2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, ormulticyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or 7membered ring containing at least one heteroatom independently selectedfrom the group consisting of O, N, and S where the ring is saturated orunsaturated, but not aromatic. The 3 or 4 membered ring contains 1heteroatom selected from the group consisting of O, N and S. The 5membered ring can contain zero or one double bond and one, two or threeheteroatoms selected from the group consisting of O, N and S. The 6 or 7membered ring contains zero, one or two double bonds and one, two orthree heteroatoms selected from the group consisting of O, N and S. Themonocyclic heterocycle is connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within themonocyclic heterocycle. Representative examples of monocyclicheterocycle include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl,piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, andtrithianyl. The bicyclic heterocycle is a monocyclic heterocycle fusedto either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl,a monocyclic heterocycle, or a monocyclic heteroaryl. The bicyclicheterocycle is connected to the parent molecular moiety through anycarbon atom or any nitrogen atom contained within the monocyclicheterocycle portion of the bicyclic ring system. Representative examplesof bicyclic heterocyclyls include, but are not limited to,2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl,indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl,decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, andoctahydrobenzofuranyl. Heterocyclyl groups are optionally substitutedwith one or two groups which are independently oxo or thia. In certainembodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclicheterocyclyl ring fused to phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the bicyclic heterocyclyl is optionally substituted by one ortwo groups which are independently oxo or thia. Multicyclic heterocyclylring systems are a monocyclic heterocyclyl ring (base ring) fused toeither (i) one ring system selected from the group consisting of abicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ringssystems independently selected from the group consisting of a phenyl, abicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic orbicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and amonocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl isattached to the parent molecular moiety through any carbon atom ornitrogen atom contained within the base ring. In certain embodiments,multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring(base ring) fused to either (i) one ring system selected from the groupconsisting of a bicyclic aryl, a bicyclic heteroaryl, a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other rings systems independently selected from the groupconsisting of a phenyl, a monocyclic heteroaryl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.Examples of multicyclic heterocyclyl groups include, but are not limitedto 10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl,9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl,10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl,1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl,12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

The term “nitro” as used herein, means a —NO₂ group.

The term “oxo” as used herein means a ═O group.

The term “saturated” as used herein means the referenced chemicalstructure does not contain any multiple carbon-carbon bonds. Forexample, a saturated cycloalkyl group as defined herein includescyclohexyl, cyclopropyl, and the like.

The term “thia” as used herein means a ═S group.

The term “unsaturated” as used herein means the referenced chemicalstructure contains at least one multiple carbon-carbon bond, but is notaromatic. For example, an unsaturated cycloalkyl group as defined hereinincludes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

The compounds of this invention may contain one or more asymmetriccarbon atoms, so that the compounds can exist in differentstereoisomeric forms. These compounds can be, for example, racemates,chiral non-racemic or diastereomers. In these situations, the singleenantiomers, i.e., optically active forms, can be obtained by asymmetricsynthesis or by resolution of the racemates. Resolution of the racematescan be accomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent; chromatography,using, for example a chiral HPLC column; or derivatizing the racemicmixture with a resolving reagent to generate diastereomers, separatingthe diastereomers via chromatography, and removing the resolving agentto generate the original compound in enantiomerically enriched form. Anyof the above procedures can be repeated to increase the enantiomericpurity of a compound.

When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless otherwise specified, itis intended that the compounds include the cis, trans, Z- andE-configurations. Likewise, all tautomeric forms are also intended to beincluded.

As used herein, the phrase “pharmaceutically acceptable salt” refers toboth pharmaceutically acceptable acid and base addition salts andsolvates. Such pharmaceutically acceptable salts include salts of acidssuch as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic,formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric,tartaric, maleic, hydroiodic, alkanoic such as acetic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Non-toxicpharmaceutical base addition salts include salts of bases such assodium, potassium, calcium, ammonium, and the like. Those skilled in theart will recognize a wide variety of non-toxic pharmaceuticallyacceptable addition salts.

As used herein, the term “subject”, “individual,” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, birds, swine, horses,livestock (e.g., pigs, sheep, goats, cattle), primates or humans.

As used here, a subject “in need thereof” refers to a subject that hasthe disorder or disease to be treated or is predisposed to or otherwiseat risk of developing the disease or disorder.

As used here, the terms “treatment” and “treating” means:

(i) inhibiting the progression the disease;

(ii) prophylactic use for example, preventing or limiting development ofa disease, condition or disorder in an individual who may be predisposedor otherwise at risk to the disease, condition or disorder but does notyet experience or display the pathology or symptomatology of thedisease;(iii) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder;(iv) ameliorating the referenced disease state, for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing or improving thepathology and/or symptomatology) such as decreasing the severity ofdisease; or(v) eliciting the referenced biological effect.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing: (1) preventing the disease; for example, preventing adisease, condition or disorder in an individual who may be predisposedto the disease, condition or disorder but does not yet experience ordisplay the pathology or symptomatology of the disease; (2) inhibitingthe disease; for example, inhibiting a disease, condition or disorder inan individual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder; and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

EXAMPLES

The methods of the disclosure are illustrated further by the followingexamples, which are not to be construed as limiting the disclosure inscope or spirit to the specific procedures and in them. It is understoodthat the nature of the substituents required for the desired targetcompound often determines the preferred method of synthesis.

General Synthetic Procedures

All chemicals were purchased from commercial suppliers and used withoutfurther purification unless otherwise stated. Reactions were monitoredwith thin-layer chromatography using silica gel 60 F254 coated glassplates (EM Sciences). Compound purification was performed with anIntelliFlash 280 automated flash chromatography system using pre-packedVarian Super Flash silica gel columns (hexanes/EtOAc or CH₂Cl₂/MeOHgradient solvent systems). A Varian Dynamax Microsorb 100-5 C₁₈ column(250 mm×21.4 mm), eluting with H₂O/CH₃CN or H₂O/MeOH gradient solventsystems (+0.05% TFA) was used for preparatory HPLC purification.Products were detected by UV at λ=254 nm, with all final compoundsdisplaying >95% purity. NMR spectra were recorded on Bruker 300 or 500MHz spectrometers at ambient temperature. Chemical shifts are reportedin parts per million (δ) and coupling constants in Hz. ¹H-NMR spectrawere referenced to the residual solvent peaks as internal standards(7.26 ppm for CDCl₃, 2.50 ppm for d₆-DMSO, and 3.34 ppm for CD₃OD). Massspectra were recorded with a Bruker Esquire Liquid Chromatograph—IonTrap Mass Spectrometer.

In some examples and embodiments, the methods useful in synthesis of theinhibitors of disclosure have been previously described in InternationalPatent Publication WO 2011/0094628 and the U.S. Patent Publication2013/0018040, both incorporated herein by reference in their entirety.

Methods of Preparation

The exemplary synthetic routes described below can be used to generatederivatives that contain varying substituents at the 1- and 3-positionsof the pyrazolopyrimidine or imidazopyrazine core.

General R₂ Alkylation Procedure:

Pyrazolopyrimidine (1 equiv.), K₂CO₃, Cs₂CO₃ or K₂CO₃:NaH₂PO₄ (1.5-2equiv.), and an alkylhalide (1.1 equiv.) or alkylmesylate (1.1 equiv.)were stirred in dry DMF at room temperature or 80° C. The reaction wasmonitored by thin layer chromatography. After completion, ethyl acetateand water were added and the organic phase was separated. The waterphase was extracted with ethyl acetate. The combined organic phases werewashed with brine, dried over Na₂SO₄ and evaporated under reducedpressure. The crude product was then purified via flash chromatographyover silica, eluting with either a hexanes/EtOAc or CH₂Cl₂/MeOHgradient. If necessary, further purification was performed withpreparatory RP-HPLC.

General Suzuki Coupling Procedure:

3-Iodopyrazolopyrimidines or 3-Bromopyrazolopyrimidines (1 equiv.),Na₂CO₃/K₃PO₄ (2-4 equiv.), Pd(PPh₃)₄/Pd(II)Cl₂dppf.DCM, (0.05 equiv.),and boronic acids or boronate pinacol esters (1-2 equiv.) were dissolvedin a mixture of dimethoxyethane (1.5 mL) and water (0.5 mL) and thenheated in a microwave at 80° C. for one hour. After cooling, ethylacetate and water were added and the organic phase was separated. Thewater phase was extracted with ethyl acetate. The combined organicphases were washed with brine, dried over Na₂SO₄ and evaporated underreduced pressure. The crude product was then purified via flashchromatography over silica, eluting with either a hexanes/EtOAc orCH₂Cl₂/MeOH gradient. If necessary, further purification was performedwith preparatory RP-HPLC.

General Naphthol Alkylation Procedure:

6-Hydroxy-2-naphthalene pyrazolopyrimidines (1 equiv.), K₂CO₃/CS₂CO₃ or(1.5-2 equiv.), and alkyl halides/epoxides (1.1 equiv.), NaH₂PO₄: K₂CO₃(1:1 equiv.), were stirred in dry DMF at room temperature or 60-80° C.and monitored by thin layer chromatography. After completion, ethylacetate and water were added and the organic phase was separated. Thewater phase was extracted with ethyl acetate. The combined organicphases were washed with brine, dried over Na₂SO₄ and evaporated underreduced pressure. The crude product was then purified via flashchromatography over silica, eluting with either a hexanes/EtOAc orCH₂Cl₂/MeOH gradient. If necessary, further purification was performedwith preparatory RP-HPLC.

General Boc-Deprotection Procedure:

Boc-amine-containing pyrazolopyrimidines was stirred in a TFA/CH₂Cl₂(1:1) mixture for 3 h. The reaction was then concentrated and purifiedvia preparatory RP-HPLC. After HPLC purification, the product was thenre-concentrated from 1.25 M HCl in EtOH to afford the final, purifiedproduct as a bis-HCl salt.

General Reductive Amination Procedure:

Deprotected pyrazolopyrimidines (1 equiv.) were dissolved in methanoland neutralized with sodium methoxide. A solution containing 2% aceticacid and an aldehyde or ketone (5-10 equiv.) was stirred at roomtemperature for 10 min. Sodium cyanoborohydride (5 equiv.) was thenadded and the reaction was stirred until reaching completion, asdetermined by thin layer chromatography (typically ˜2 h). The reactioncrude was then purified via preparatory RP-HPLC. After HPLCpurification, the residue was dissolved in a small amount of 2 M HCl inmethanol and, after concentration in vacuo, the final product wasobtained as an HCl salt.

General Pinacol Ester Formation Procedure:

Alkylated naphthols or quinolones (1 equiv.), Cs₂CO₃ (1.5-2 equiv.),pinacolatodiborane (2.0 equiv.), Pd(II)Cl₂(dppf).DCM (0.05 equiv.), andKOAc (1 equiv.) in dry DMSO were heated at 85° C. for 5-8 h. Aftercompletion, ethyl acetate and water were added and the organic phase wasseparated. The water phase was extracted with ethyl acetate. Thecombined organic phases were washed with brine, dried over Na₂SO₄ andevaporated under reduced pressure. The crude product was then purifiedvia flash chromatography over silica, eluting with a hexanes/EtOAcsolvent gradient.

General Procedure for Boronylation Using Triisopropylborate:

Aryl halides (1 equiv.) and triisopropylborate (1.5 equiv.) weredissolved in tetrahydrofuran:toluene (2:8), cooled to −78° C., andn-Buli (1.7 equiv.) was added dropwise over 30-40 min. After addition,the reaction was stirred at −78° C. for 1 h. After 1 h, the reaction wasallowed to warm to 0° C. and stirred for 15-25 min followed by additionof 2N HCl slowly. The organic layer was separated and concentrated invacuo to afford the desired crude product as a white crystalline productor by collecting and washing with water the white crystalline solid thatforms upon addition of 2N HCl.

Example 1:3-(6-Cyclobutoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

2-Bromo-6-cyclobutoxynaphthalene: 6-bromonaphthalene (700 mg, 3.1 mmol),K₂CO₃ (2.140 g, 15.5 mmol), and bromocyclobutane (1.75 mL, 18.6 mmol) indry DMF were heated at 180° C. in a microwave for 8 h. After completion,ethyl acetate and water were added and the organic phase was separated.The aqueous phase was extracted with ethyl acetate (2×10 mL). Thecombined organic phases were washed with brine, dried over Na₂SO₄ andevaporated under reduced pressure. The crude product was then purifiedvia flash chromatography over silica, eluting with a hexanes/EtOAcsolvent gradient to afford 693 mg (80% yield) of pure product. ¹H NMR(300 MHz, CDCl₃) δ7.88 (s, 1H), 7.65-7.43 (m, 3H), 7.10 (dd, 1H), 6.94(s, 1H), 4.74 (m, 1H), 2.58-2.45 (m, 2H), 2.30-2.13 (m, 2H), 1.95-1.69(m, 2H); MS (ESI) 278.5 m/z [MH⁺], C₁₄H₁₄BrO requires 278.2.

2-(6-Cycl obutoxynaphth alen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane:2-Bromo-6-cyclobutoxynaphthalene and pinacolatodiborane were subjectedto general pinacol ester formation procedure to afford the desired pureproduct (631 mg, 60% yield); ¹H NMR (300 MHz, CDCl₃) δ 8.26 (s, 1H),7.93-7.61 (m, 3H), 7.2-6.93 (m, 2H), 4.80 (m, 1H), 2.67-2.36 (m, 2H),2.41-2.11 (m, 2H), 2.01-1.60 (m, 2H), 1.37 (s, 12H); MS (ESI) 325.1 m/z[MH⁺], C₂₀H₂₆BO₃ requires 325.1.

2-(6-Cyclobutoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand tert-butyl4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylatewere subjected to the general Suzuki coupling procedure followed by thegeneral boc-deprotection procedure and general reductive aminationprocedure in order to afford3-(6-cyclobutoxy-naphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CD₃OD) δ8.47 (s, 1H), 8.15 (s, 1H), 7.99 (d, J=8.3 Hz,1H), 7.92 (d, J=9.5 Hz, 1H), 7.80 (d, J=8.9 Hz, 1H), 7.24 (m, 2H), 4.53(d, J=6.6 Hz, 2H), 3.56 (m, 2H), 3.03 (m, 2H), 2.86 (s, 3H), 2.62 (m,2H), 2.44 (m, 1H), 2.22 (m, 2H), 2.04-1.60 (m, 6H); MS (ESI) 443.4 m/z[MH+], C₂₆H₃₁N₆O requires 443.2.

Example 2:3-(4-Amino-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol

6-Bromo-2-(2,2,2-trifluoroethoxy)quinolone: 6-bromo-2-chloroquinoline(1.00 g, 4.1 mmol), CF₃CH₂OH (0.95 mL, 12.3 mmol), N-Methylmorpholine(12 mL), and NaOH (330 mg, 8.2 mmol) were taken in microwave tube andthen heated at 165° C. for 45 min. After addition of water, the reactionmixture was extracted with ethyl acetate (3×20 mL). The organic layerwas washed with brine, dried over sodium sulfate, and evaporated underreduced pressure. The crude compound was then taken to the next stepwithout further purification.

Synthesis of 2-(2,2,2-Trifluoroethoxy)quinolin-6-ylboronic acid:6-Bromo-2-(2,2,2-trifluoroethoxy)quinoline was subjected to the generalprocedure for boronylation using triisopropylborate to afford a whitecrystalline product (354 mg, 80% yield,). ¹H NMR (300 MHz, CD₃OD) δ6.91(s, 1H), 6.82 (d, J=8.5 Hz, 1H,), 6.45 (s, 2H) 6.61 (d, J=7.04 Hz, 1H),3.5 (q, J=8.7 Hz, 2H,); MS (ESI) 272.4 m/z [MH+], C₁₁H₁₀BF₃NO₃ requires272.2.

2-(2,2,2-Trifluoroethoxy)quinolin-6-ylboronic acid and3-(4-amino-3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-olwere subjected to the general Suzuki coupling procedure in order toafford3-(4-Amino-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol.¹H NMR (300 MHz, CD₃OD) δ8.40 (s, 1H), 8.17 (d, J=8.7 Hz, 1H), 8.07 (d,J=1.6 Hz, 1H), 8.01-7.95 (m, 2H), 7.12 (d, J=8.9 Hz, 1H), 4.98 (q, J=8.5Hz, 2H), 4.35 (s, 2H), 3.15 (s, 2H), 1.05 (s, 6H); MS (ESI) 447.5 m/z[MH+], C₂₁H₂₁F₃N₆O₂ requires 447.4.

Example 3:3-(2-Cyclopropoxyquinolin-6-yl)-4-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

6-Bromo-2-cyclopropoxyquinoline: 6-Bromo-quinolin-2(1H)-one (1.00 g, 4.4mmol, 1 equiv.), Cs₂CO₃ (5.08 g, 17.8 mmol), and bromocyclopropane (1.06g, 13.3 mmol) in dry DMF (10 mL) were heated at 180° C. in a microwavefor 3 h. After completion, ethyl acetate and water were added and theorganic phase was separated. The water phase was further extracted withethyl acetate. The combined organic phases were washed with brine, driedover Na₂SO₄ and evaporated under reduced pressure. The crude product wasthen purified via flash chromatography over silica, eluting with ahexanes/EtOAc solvent gradient to afford 0.235 mg (20% yield) of pureproduct. ¹H NMR (300 MHz, CDCl₃) δ7.89 (s, 1H), 7.87-7.82 (m, 1H),7.78-7.65 (m, 2H), 6.88 (d, J=8.91 Hz, 1H), 4.54-4.44 (m, 1H), 0.94-0.77(m, 4H); MS (ESI) 265.5 m/z [MH⁺], C₁₂H₁₁BrNO requires 265.2.

2-Cyclopropoxyquinolin-6-ylboronic acid: 6-Bromo-2-cyclopropoxyquinoline(2.01 g, 7.95 mmol, 1 equiv.) and triisopropylborate (2.05 mg, 13.5mmol, 1.69 equiv.) were subjected to general procedure for boronylationusing triisopropylborate to afford the desired pure product (1.05 g, 80%yield); ¹H NMR (300 MHz, DMSO) δ8.37-8.28 (m, 2H), 8.10-8.04 (m, 1H),7.77 (d, J=8.5 Hz, 1H), 7.06 (d, J=8.9 Hz, 1H), 4.55-4.45 (m, 1H),0.91-0.81 (m, 2H), 0.80-0.73 (m, 2H); MS (ESI) 230.2 m/z [MH⁺],C₁₂H₁₃BNO₃ requires 230.2.

2-Cyclopropoxyquinolin-6-ylboronic acid and tert-butyl4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylatewere subjected to the general Suzuki coupling procedure followed by thegeneral boc-deprotection procedure and general reductive aminationprocedure in order to afford3-(2-cyclopropoxyquinolin-6-yl)-14(1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CD₃OD) δ9.17 (s, 1H), 8.75-8.33 (m, 3H), 8.28-7.98 (m,2H), 4.72 (m, 1H), 4.54 (d, J=6.0 Hz, 2H), 3.56 (m, 2H), 3.10 (m, 2H),3.00 (s, 3H), 2.48 (m, 1H), 2.00 (m, 2H), 1.60 (m, 2H), 1.20-1.10 (m,4H); MS (ESI) 430.5 m/z [MH+], C₂₄H₂₈N₇O requires 430.6.

Example 4:3-(6-Cyclopropoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

2-Bromo-6-cyclopropoxynaphthalene: 6-Bromonaphthalen-2-ol (3.00 g, 13.0mmol), Cs₂CO₃ (1.29 g, 39.6 mmol) and bromocyclopropane (4.07 g, 39.0mmol) were taken in a microwave tube and heated at 180° C. for 30 min.After completion, ethyl acetate and water were added and the organicphase was separated. The water phase was further extracted with ethylacetate. The combined organic phases were washed with brine, dried overNa₂SO₄ and evaporated under reduced pressure. The crude product was thenpurified via flash chromatography over silica, eluting with ahexanes/EtOAc solvent gradient to afford 2.50 g (71% yield) of pureproduct. ¹H NMR (300 MHz, CDCl₃): δ ppm 7.91 (m, 1H), 7.66-7.58 (dd,J=8.9, 4.6 Hz, 2H), 7.53-7.46 (dd, J=8.7, 1.9 Hz, 1H), 7.39 (d, J=2.1Hz, 1H), 7.18-7.12 (dd, J=8.9, 2.3 Hz, 1H), 3.83 (m, 1H), 0.87-0.78 (m,4H); MS (ESI): 264.2 m/z [MH+], C₁₃H₁₂BrO requires 264.2.

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane:2-Bromo-6-cyclopropoxynaphthalene was subjected to the general pinacolester formation procedure to afford 1.01 g, (65% yield) of a whitecrystalline product. ¹H NMR (300 MHz, CDCl₃): δ ppm 8.27 (s, 1H), 7.77(m, 3H) 7.45 (d, J=2.3 Hz, 1H), 7.14 (dd, J=8.9, 2.48 Hz, 1H), 3.88 (m,1H), 1.40 (s, 12H), 0.87 (m, 2H), 0.82 (m, 2H); MS (ESI): 311.5 m/z[MH+], C₁₉H₂₄BO₃ requires 311.2.

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand tert-butyl4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylatewere subjected to the general Suzuki coupling procedure followed by thegeneral boc-deprotection procedure and general reductive aminationprocedure in order to afford3-(6-cyclopropoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CD₃OD) δ8.49 (s, 1H), 8.17 (s, 1H), 8.03 (d, J=8.5 Hz,1H), 7.92 (d, J=9.1 Hz, 1H), 7.80 (d, J=8.5 Hz, 1H), 7.64 (d, J=2.0 Hz,1H), 7.27 (dd, J=8.9, 1.8 Hz, 1H), 4.52 (d, J=6.0 Hz, 2H), 3.98 (m, 1H),3.56 (m, 2H), 3.05 (m, 2H), 2.87 (s, 3H), 2.44 (m, 1H), 2.00 (m, 2H),1.76 (m, 2H), 0.92 (m, 2H), 0.80 (m, 2H); MS (ESI) 429.5 m/z [MH+],C₂₅H₂₉N₆O requires 429.6.

Example 5:3-(2-Cyclopropoxyquinolin-6-yl)-1-(3-(dimethylamino)-2,2-dimethylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

1-(3-(Dimethylamino)-2,2-dimethylpropyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-aminewas generated with 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine and3-bromo-N,N,2,2-tetramethylpropan-1-amine using the general R₂alkylation procedure, to afford the product as pale yellow solid, (286mg, 40% yield); ¹H NMR (300 MHz, MeOD4) δ 8.38 (s, 1H), 4.46 (s, 2H),3.22 (s, 2H), 3.06 (s, 6H), 1.16 (s, 6H); MS (ESI) 375.2 [MH+],C₁₂H₂₀IN₆ requires 375.2.

2-Cyclopropoxyquinolin-6-ylboronic acid and1-(3-(dimethylamino)-2,2-dimethylpropyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere subjected to the general Suzuki coupling procedure to afford3-(2-cyclopropoxyquinolin-6-yl)-1-(3-(dimethylamino)-2,2-dimethylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CD₃OD) δ8.29 (s, 1H), 8.22 (d, J=8.9 Hz, 1H,), 8.11 (d,J=1.6 Hz, 1H) 8.01 (s, 1H), 8.00 (dd, J=8.5, 1.5 Hz, 1H,), 7.07 (d,J=8.9 Hz, 1H), 4.46 (m, 1H), 4.36 (s, 2H), 2.46 (s, 2H), 2.44 (s, 6H),1.03 (s, 6H), 0.93-0.82 (m, 4H). MS (ESI) 432.6 m/z [MH+], C₂₄H₃₀N₇Orequires 432.5.

Example 6:1-(4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

1-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olwas generating using the general R₂ alkylation procedure using3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (500 mg, 1.92 mmol),2,2-dimethyloxirane (0.276 mg, 3.80 mmol), and K₂CO₃:NaH₂PO₄ (0.262 mg,1.90 mmol) in 3 mL of a acetonitrile:water (8.5:1.5) mixture. Thereaction was stirred at 150° C. for 3 h in a microwave, affording1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol(150 mg, yield, 23.4% yield) as a white solid after purification using amethanol/dichloromethane solvent gradient. ¹H NMR (300 MHz, CD₃OD) δ8.26(s, 1H), 4.37 (s, 2H), 1.29 (s, 6H). MS (ESI) m/z 334.2 [MH⁺], C₉H₁₃IN₅Orequires 334.1.

6-ethoxynaphthalen-2-ylboronic acid and1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olwere subjected to the general Suzuki coupling procedure to affordcompound1-(4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol;¹H NMR (300 MHz, CD₃OD) δ8.26 (s, 1H), 8.09 (s, 1H), 7.97-7.83 (m, 2H),7.76 (dd, J=8.2, 1.4 Hz, 1H), 7.30 (d, J=2.0 Hz, 1H), 7.20 (dd, J=8.9,1.4 Hz, 1H), 4.41 (s, 2H), 4.19 (q, J=7.0 Hz, 2H), 1.47 (t, J=6.8 Hz,3H), 1.28 (s, 6H). MS (ESI) 378.2 m/z [MH+], C₂₁H₂₄N₅O₂ requires 378.1.

Example 7:3-(6-cyclopropoxynaphthalen-2-yl)-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Methanesulfonyl chloride (7.54 mL, 97.9 mmol) was added slowly at 0° C.to a solution of (3-methyloxetan-3-yl)methanol (5.00 mg, 48.9 mmol) andtriethylamine (13 mL, 97 mmol) in dichloromethane (20 mL). The reactionwas stirred for 5 h at room temperature. After completion of thereaction, dichloromethane was removed by reduced pressure and thereaction was diluted with ethyl acetate. The ethyl acetate was washedwith NaHCO₃ (25 mL), 1N HCl, brine (50 mL), dried over sodium sulfate,and concentrated. The crude product was subjected to flashchromatography using a hexane/ethyl acetate solvent gradient to affordpure 3-methyloxetan-3-yl)methyl methanesulfonate (4.40 g, 50% yield). ¹HNMR (301 MHz, CDCl₃) δ4.70-4.12 (m, 6H), 3.07 (s, 3H), 1.39 (s, 3H); MS(ESI) 181.1 [MH+], C₆H₁₃O₄S requires 181.0.

3-Iodo-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-aminewas generated by subjecting 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine(150 mg, 0.60 mmol) and (3-methyloxetan-3-yl)methyl methanesulfonate (83mg, 0.46 mmol) to the general R₂ alkylation procedure (110 mg, 55%yield). ¹H NMR (300 MHz, CDCl₃) δ8.33 (s, 1H), 5.94 (s, 2H), 4.79 (d,J=6.4 Hz, 2H), 4.56 (s, 2H), 4.41 (d, J=6.1 Hz, 2H), 1.28 (s, 3H); MS(ESI) m/z 346.2 [MH⁺], C₁₀H₁₃IN₅O requires 346.1.

2-(6-cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand3-iodo-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere subjected to the general Suzuki coupling procedure in order toafford3-(6-cyclopropoxynaphthalen-2-yl)-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CDCl₃) δ8.39 (s, 1H), 8.08 (s, 1H), 7.93 (d, J=8.7 Hz,1H), 7.83 (d, J=8.9 Hz, 1H), 7.77 (d, J=9.4 Hz, 1H), 7.52 (s, 1H), 7.24(dd, J=10.0, 3.0 Hz, 1H), 5.68 (s, 2H), 4.90 (d, J=6.1 Hz, 2H), 4.64 (s,2H), 4.45 (d, J=6.1 Hz, 2H), 3.91 (m, 1H), 1.36 (3, 3H), 0.96-0.83 (m,4H); MS (ESI) 402.2 m/z [MH+], C₂₃H₂₄N₅O₂ requires 402.4.

Example 8:2-((4-Amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-ol

2-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-olwas generated by subjecting 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine(250 mg, 0.96 mmol), 2,2-bis(trifluoromethyl)oxirane (0.17 mL, 1.44mmol), and K₂CO₃:NaH₂PO₄ (198 mg, 1.44 mmol) to the general R₂alkylation procedure (126 mg, 30% yield). ¹H-NMR (301 MHz, CDCl₃) δ8.33(s, 1H), 8.02 (s, 1H), 6.33 (s, 2H); MS (ESI) m/z 442.1 [MH⁺],C₉H₆F₆IN₅O requires 442.2.

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand2-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-olwere subjected to the general Suzuki coupling procedure in order toafford2-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-ol.¹H NMR (300 MHz, CD₃OD) δ8.32 (s, 1H), 8.11 (s, 1H), 7.97 (d, J=8.5 Hz,1H), 7.87 (d, J=9.1 Hz, 1H), 7.76 (dd, J=8.5, 1.6 Hz, 1H), 7.59 (d, 1H),7.23 (dd, J=8.9, 2.2 Hz, 1H), 5.03 (s, 2H), 3.94 (m, 1H), 0.94-0.76 (m,4H); MS (ESI) 498.2 m/z [MH+], C₂₂H₁₈F₆N₅O₂ requires 498.2.

Example 9:3-(6-cyclopropoxynaphthalen-2-yl)-1-isobutyl-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

6-tert-butyldimethylsilyloxy-2-naphthaleneboronic acid and3-iodo-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine² were subjected tothe general Suzuki coupling procedure. The crude product was purified bysilica gel using dichloromethane/methanol gradient (note: deprotectionof the tert-butyldimethylsilyloxy protecting group was observed afterpurification). ¹H NMR (300 MHz, CD₃OD) δ8.27 (s, 1H), 8.03 (s, 1H), 7.82(m, 2H), 7.71 (m, 1H), 7.18 (m, 2H), 4.21 (d, J=4.2 Hz, 2H), 2.39 (m,1H), 0.96 (d, J=6.5 Hz, 6H); MS (ESI) 334.4 m/z [MH+], C₁₉H₁₉N₅Orequires 334.2.

6-(4-amino-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-oland bromocyclopropane were subjected to the general naphthol alkylationprocedure in order to afford3-(6-cyclopropoxynaphthalen-2-yl)-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine.¹H NMR (300 MHz, CD₃OD) δ8.27 (s, 1H), 8.10 (s, 1H), 7.98 (d, J=8.7 Hz,1H), 7.89 (d, J=8.9 Hz, 1H), 7.76 (dd, J=8.5, 1.6 Hz, 1H), 7.61 (d,J=2.2 Hz, 1H), 7.25-7.18 (dd, J=8.9, 2.2 Hz, 1H), 4.24 (d, J=7.25 Hz,2H), 3.95 (m, 1H), 2.38 (m, 1H), 0.97 (d, J=6.6 Hz, 6H), 0.90 (m, 2H),0.79 (m, 2H); MS (ESI) 374.2 m/z [MH+], C₂₂H₂₄N₅O requires 374.4.

Example 10:3-(6-(Cyclopropylmethoxy)naphthalen-2-yl)-1-((1-methylpiperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Tert-butyl4-((4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylateand (bromomethyl)cyclopropane were subjected to the general naphtholalkylation procedure followed by the general boc-deprotection procedureand general reductive amination procedure in order to afford theproduct. ¹H NMR (300 MHz, CD₃OD) δ8.51 (s, 1H), 8.17 (s, 1H), 8.01 (d,J=8.5 Hz, 1H), 7.93 (d, J=9.1 Hz, 1H), 7.81 (d, J=9.3 Hz, 1H), 7.36 (s,1H), 7.30 (d, J=9.3 Hz, 1H), 4.56 (d, J=6.4 Hz, 2H), 4.03 (d, J=6.6 Hz,2H), 3.57 (m, 2H), 3.06 (m, 2H), 2.88 (s, 3H), 2.45 (m, 1H), 2.02 (m,2H), 1.77 (m, 2H), 1.44 (m, 1H), 0.71 (m, 2H), 0.46 (m, 2H); MS (ESI)443.5 m/z [MH+], C₂₆H₃₁N₆O requires 443.5.

Example 11:3-(6-(2-Methoxyethoxy)naphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Tert-butyl4-((4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylateand bromomethoxyethane were subjected to the general naphthol alkylationprocedure followed by the general boc-deprotection procedure and generalreductive amination procedure in order to afford the product. ¹H NMR(300 MHz, CD₃OD) δ8.50 (s, 1H), 8.16 (s, 1H), 8.01 (d, J=7.8 Hz, 1H),7.93 (d, J=8.5 Hz, 1H), 7.80 (d, J=8.3 Hz, 1H), 7.40 (s, 1H), 7.30 (d,J=7.8 Hz, 1H), 4.52 (d, J=4.2 Hz, 2H), 4.31 (m, 2H), 3.86 (m, 2H), 3.62(m, 2H), 3.54 (s, 3H), 3.04 (m, 2H), 2.87 (s, 3H), 2.44 (m, 1H), 1.99(m, 2H), 1.77 (m, 2H); MS (ESI) 447.5 m/z [MH+], C₂₅H₃₁N₆O₂ requires447.5.

Example 12:1-((1-Methylpiperidin-4-yl)methyl)-3-(6-(oxetan-3-yloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

Tert-butyl4-((4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylateand 3-bromooxetane were subjected to the general naphthol alkylationprocedure followed by the general boc-deprotection procedure and generalreductive amination procedure in order to afford the product. ¹H NMR(300 MHz, CD₃OD) δ8.49 (s, 1H), 8.18 (s, 1H), 8.06-7.92 (m, 2H), 7.82(d, J=8.5 Hz, 1H), 7.51 (s, 1H), 7.36 (d, J=9.1 Hz, 1H), 4.81 (m, 1H),4.53 (d, J=5.6 Hz, 2H), 3.99-3.84 (m, 4H), 3.57 (m, 2H), 3.05 (m, 2H),2.86 (s, 3H), 2.44 (m, 1H), 2.00 (m, 2H), 1.74 (m, 2H); MS (ESI) 445.2m/z [MH+], C₂₅H₂₉N₆O₂ requires 445.2.

Example 13:2-(6-(4-Amino-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yloxy)ethanol

Tert-butyl4-((4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylateand oxirane were subjected to the general naphthol alkylation procedurefollowed by the general boc-deprotection procedure and general reductiveamination procedure in order to afford the product. ¹H NMR (300 MHz,CD₃OD) δ8.50 (s, 1H), 8.17 (s, 1H), 8.02 (d, J=8.3 Hz, 1H), 7.94 (d,J=8.9 Hz, 1H), 7.80 (d, J=8.9 Hz, 1H), 7.41 (d, J=2.2 Hz, 1H), 7.34 (dd,J=8.7, 2.0 Hz, 1H), 4.53 (d, J=6.4 Hz, 2H), 4.25 (t, J=4.5 Hz, 2H), 3.99(t, J=4.5 Hz, 2H), 3.56 (m, 2H), 3.04 (m, 2H), 2.86 (s, 3H), 2.44 (m,1H), 2.00 (m, 2H), 1.72 (m, 2H); MS (ESI) 433.3 m/z [MH+], C₂₄H₂₉N₆O₂requires 433.5.

Example 14:1-(6-(4-Amino-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yloxy)-2-methylpropan-2-ol

Tert-butyl4-((4-amino-3-(6-hydroxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylateand 2,2-dimethyloxirane were subjected to the general naphtholalkylation procedure followed by the general boc-deprotection procedureand general reductive amination procedure in order to afford theproduct. ¹H NMR (300 MHz, CD₃OD) δ8.48 (s, 1H), 8.17 (s, 1H), 8.02 (d,J=8.9 Hz, 1H), 7.94 (d, J=8.7 Hz, 1H), 7.79 (d, J=8.5 Hz, 1H), 7.43-7.32(m, 2H), 4.53 (d, J=6.8 Hz, 2H), 3.98 (s, 2H), 3.56 (m, 2H), 3.04 (m,2H), 2.87 (s, 3H), 2.44 (m, 1H), 2.00 (m, 2H), 1.77 (m, 2H), 1.41 (s,6H); MS (ESI) 461.5 m/z [MH+], C₂₆H₃₃N₆O₂ requires 461.5.

Example 15:3-(2-Ethoxyquinolin-6-yl)-14(1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

Tert-butyl4-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylatewas subjected to the general boc-deprotection procedure and generalreductive amination procedure in order to afford the product. ¹H NMR(300 MHz, CD₃OD) δ8.29 (s, 1H), 8.22 (d, J=8.2 Hz, 1H), 8.12 (s, 1H),7.98 (m, 2H), 7.04 (d, J=8.5 Hz, 1H), 4.55 (q, J=6.8 Hz, 2H), 4.38 (d,J=4.3 Hz, 2H), 2.95 (m, 2H), 2.30 (s, 3H), 2.99 (m, 2H),1.67 (m, 2H),1.48 (m, 4H); MS (ESI) 418.3 m/z [MH+], C₂₃H₂₈N₇O requires 418.5.

Example 16:1-((1-Methylpiperidin-4-yl)methyl)-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

2-(2,2,2-Trifluoroethoxy)quinolin-6-ylboronic acid and tert-butyl4-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylatewere subjected to the general Suzuki coupling procedure followed by thegeneral boc-deprotection procedure and general reductive aminationprocedure in order to afford the product. ¹H NMR (300 MHz, CD₃OD) δ8.49(s, 1H), 8.39 (d, J=8.5 Hz, 1H), 8.24 (s, 1H), 8.04 (s, 2H), 7.18 (d,J=7.0 Hz, 1H), 5.09 (q, J=8.7 Hz, 2H), 4.52 (s, 2H), 3.54 (m, 2H), 3.04(m, 2H), 2.84 (s, 3H), 2.45 (m, 1H), 1.98 (m, 2H), 1.75 (m, 2H); MS(ESI) 472.2 m/z [MH+], C₂₃H₂₅F₃N₇O requires 472.5.

Example 17:3-(6-ethoxynaphthalen-2-yl)-1-isobutyl-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

6-ethoxynaphthalen-2-ylboronic acid and3-iodo-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine were subjected tothe general Suzuki coupling procedure in order to afford the product. ¹HNMR (300 MHz, CD₃OD) δ8.27 (s, 1H), 8.09 (s, 1H), 7.96 (d, J=8.1 Hz,1H), 7.89 (d, J=9.1 Hz, 1H), 7.75 (dd, J=8.2, 2.0 Hz, 1H), 7.32 (s, 1H),7.22 (dd, 2.4 Hz, 1H), 4.28-4.17 (m, 4H), 2.39 (m, 1H), 1.48 (t, J=6.8Hz, 3H), 0.96 (d, J=6.6 Hz, 6H); MS (ESI) 362.4 m/z [MH+], C₂₁H₂₄N₅Orequires 362.2.

Example 18:3-(2-Cyclopropoxyquinolin-6-yl)-1-isobutyl-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

2-Cyclopropoxyquinolin-6-ylboronic acid and3-iodo-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine were subjected tothe general Suzuki coupling procedure in order to afford the product. ¹HNMR (300 MHz, CD₃OD) δ8.25 (s, 1H), 8.23 (d, J=8.9 Hz, 1H), 8.10 (s,1H), 8.00-7.91 (m, 2H), 7.02 (d, J=8.7 Hz, 1H), 4.46 (s, 1H), 4.22 (d,J=7.4 Hz, 2H), 2.36 (m, 1H), 0.96 (d, J=6.6 Hz, 6H), 0.91-0.72 (m, 4H);MS (ESI) 375.4 m/z [MH+], C₂₁H₂₃N₆O requires 375.4.

Example 19:3-(4-Amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol

2-Cyclopropoxyquinolin-6-ylboronic acid and previously reported3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-olwere subjected to the general Suzuki coupling procedure in order toafford the product. ¹H NMR (300 MHz, CD₃OD) δ8.65 (s, 1H), 8.50 (s, 1H),8.36 (m, 1H), 8.21-8.05 (m, 2H), 7.51 (d, J=8.3 Hz, 1H), 4.59 (s, 2H),4.47 (s, 2H), 4.46 (m, 1H), 1.01 (s, 6H), 0.70 (m, 4H); MS (ESI) 405.2m/z [MH+], C₂₂H₂₅N₆O₂ requires 405.4.

Example 20:3-(2-Cyclopropoxyquinolin-6-yl)-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

2-Cyclopropoxyquinolin-6-ylboronic acid and3-iodo-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere subjected to the general Suzuki coupling procedure in order toafford the product. ¹H NMR (300 MHz, CDCl₃) δ8.41 (s, 1H), 8.12-8.01 (m,3H), 7.96 (d, J=8.3 Hz, 1H), 6.97 (d, J=8.0 Hz, 1H), 5.54 (s, 2H), 4.91(d, J=6.1 Hz, 2H), 4.64 (s, 2H), 4.56 (m, 1H), 4.45 (d, J=6.1 Hz, 2H),1.36 (s, 3H), 0.95-0.82 (m, 4H); MS (ESI) 403.2 m/z [MH+], C₂₂H₂₃N₆O₂requires 403.4.

Example 21:3-(6-Cyclopropoxynaphthalen-2-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand3-iodo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere subjected to the general Suzuki coupling procedure in order toafford the product. ¹H NMR (300 MHz, CD₃OD) δ8.47 (s, 1H), 8.16 (s, 1H),8.03 (d, J=8.2 Hz, 1H), 7.92 (d, J=9.1 Hz, 1H), 7.78 (d, J=8.2 Hz, 1H),7.64 (s, 1H), 7.27 (dd, J=9.1, 2.2 Hz, 1H) 4.45 (d, J=6.8 Hz, 2H,), 3.99(m, 1H), 3.76 (m, 2H), 3.05 (m, 2H), 2.40 (m, 1H), 1.61 (m, 2H), 1.46(m, 2H), 0.93-0.80 (m, 4H); MS (ESI) 416.4 m/z [MH+], C₂₄H₂₅N₅O₂requires 416.4.

Example 22:3-(2-Cyclopropoxyquinolin-6-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-(1]pyrimidin-4-amine

2-cyclopropoxyquinolin-6-ylboronic acid and3-iodo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-aminewere subjected to the general Suzuki coupling procedure in order toafford the product. ¹H NMR (300 MHz, CD₃OD) δ8.31 (s, 1H), 8.22 (d,J=8.9 Hz, 1H), 8.12 (d, J=1.8 Hz, 1H), 8.06 (d, J=8.7 Hz, 1H), 8.00 (dd,1.2 Hz, 1H), 7.07 (d, J=8.9 Hz, 1H), 4.47 (m, 1H) 4.36 (d, J=7.2 Hz,2H,), 3.98 (m, 2H), 3.42 (m, 2H), 2.34 (m, 1H), 1.59 (m, 2H), 1.49 (m,2H), 0.93-0.83 (m, 4H); MS (ESI) 416.4 m/z [MH+], C₂₃H₂₄N₆O₂ requires416.4.

Example 23:1-(4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

2-ethoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline and1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olwere subjected to the general Suzuki coupling procedure to affordcompound the product. ¹H NMR (300 MHz, CD₃OD) δ8.69 (d, J=9.1 Hz, 1H),8.45 (s, 1H), 8.34 (s, 1H), 8.17 (d, J=8.0 Hz, 1H), 8.04 (d, J=7.4 Hz,1H), 7.45 (d, J=7.4 Hz, 1H), 4.67 (q, J=6.0 Hz, 2H), 4.50 (s, 2H), 1.54(t, J=6.5 Hz, 3H), 1.31 (s, 6H). MS (ESI) 379.2 m/z [MH+], C₂₀H₂₃N₆O₂requires 379.1.

Example 24:1-(4-Amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olwere subjected to the general Suzuki coupling procedure to afford titlecompound the product; ¹H NMR (500 MHz, CD₃OD) δ8.26 (s, 1H), 8.08 (s,1H), 7.93 (d, J=8.4 Hz, 1H), 7.85 (d, J=8.81 Hz, 1H), 7.76 (d, J=8.4 Hz,1H), 7.57 (s, 1H), 7.20 (dd, J=8.8, 1.8 Hz, 1H), 4.40 (s, 2H), 3.91 (m,1H), 1.27 (s, 6H), 0.88 (m, 2H), 0.78 (m, 2H). MS (ESI) 390.2 m/z [MH+],C₂₂H₂₄N₅O₂ requires 390.1.

Example 25:1-(4-Amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol

2-Cyclopropoxyquinolin-6-ylboronic acid and1-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olwere subjected to the general Suzuki coupling procedure in order toafford the product. NMR (300 MHz, CD₃OD) δ8.28 (s, 1H), 8.28-8.24 (d,J=8.9 Hz, 1H), 8.15 (t, J=1.4 Hz, 1H), 8.01 (d, J=1.2 Hz, 2H), 7.06 (d,J=8.7 Hz, 1H), 4.53-4.45 (m, 1H), 4.42 (s, 2H), 1.28 (s, 6H), 0.92-0.85(m, 2H), 0.85-0.77 (m, 2H); MS (ESI) 391.1 m/z [MH⁺], C₂₁H₂₃N₆O₂requires 391.2.

Example 26:3-(4-Amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol

2-(6-Cyclopropoxynaphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneand previously reported3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-olwere subjected to the general Suzuki coupling procedure in order toafford the product. ¹H NMR (300 MHz, CD₃OD) δ8.30 (s, 1H), 8.12 (s, 1H),8.00 (d, J=8.7 Hz, 1H), 7.90 (d, J=8.9 Hz, 1H), 7.78 (dd, 1.5 Hz, 1H),7.62 (d, 1H), 7.24 (dd, J=9.1, 2.4 Hz, 1H), 4.33 (s, 2H), 3.97 (m, 1H),3.32 (s, 2H), 1.22 (s, 6H), 0.94-0.81 (m, 4H); MS (ESI) 404.5 m/z [MH+],C₂₃H₂₆N₅O₂ requires 404.4.

Examples 27-119

The following compounds were prepared by the methothes disclosed herein:

Ex. No. Compound 273-(2-methoxyquinolin-6-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 283-(2-(cyclopropylmethoxy)quinolin-6-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 293-(6-(cyclopropylmethoxy)naphthalen-2-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 303-(6-cyclobutoxynaphthalen-2-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 313-(6-(oxetan-3-yloxy)naphthalen-2-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 323-(4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol 331-(6-(4-amino-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yloxy)-2-methylpropan-2-ol 342-(6-(4-amino-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yloxy)ethanol 351-isobutyl-3-(6-(oxetan-3-yloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 361-isobutyl-3-(6-(2-methoxyethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 373-(4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropan-1-ol 383-(2-(2-methoxyethoxy)quinolin-6-yl)-1-neopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 393-(2-cyclopropoxyquinolin-6-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 401-(azetidin-3-ylmethyl)-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 413-(2-cyclopropoxyquinolin-6-yl)-1-((1-methylazetidin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 423-(2-(2-methoxyethoxy)quinolin-6-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 431-isobutyl-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 441-(piperidin-4-ylmethyl)-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 452-(3-(4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropoxy)ethanol 461-(4-amino-3-(2-(2,2,2-trifluoroethoxy)quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-ol 473-(7-ethoxynaphthalen-2-yl)-1-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine48 3-(7-ethoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 493-(6-ethoxynaphthalen-2-yl)-1-((3-methyloxetan-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 502-(4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-morpholinoethanone 512-(4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-morpholinoethanone 522-((4-amino-3-(6-(methoxymethyl)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-ol 532-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1,1,1,3,3,3-hexafluoropropan-2-ol 541-(3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrrolo[3,2-c]pyridin-1-yl)-2-methylpropan-2-ol 551-(4-amino-5-(2-cyclopropoxyquinolin-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-methylpropan-2-ol 564-(4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,3,3-trimethylbutan-2-ol 571-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)cyclobutanol 581-(4-amino-5-(6-cyclopropoxynaphthalen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-methylpropan-2-ol 591-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)cyclobutanol 601-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)cyclopentanol 611-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)cyclopentanol 624-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-tetrahydro-2H-pyran-4-ol 634-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-tetrahydro-2H-pyran-4-ol 64(3-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)oxetan-3-yl)methanol 65(3-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)oxetan-3-yl)methanol 663-(6-cyclopropoxynaphthalen-2-yl)-1-(2-methoxy-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 673-(2-cyclopropoxyquinolin-6-yl)-1-(2-methoxy-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 68 methyl3-(4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropanoate 69 methyl3-(4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropanoate 704-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-4-ol 714-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 72 methyl2-((6-(4-amino-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yl)oxy)acetate 732-((6-(4-amino-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yl)oxy)acetonitrile 744-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-4-carbonitrile 754-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidine-4-carbonitrile 763-(4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2-dimethylpropanoic acid 773-(6-cyclopropoxynaphthalen-2-yl)-1-(2-fluoro-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 784-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-4-ol 794-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 804-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 814-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 824-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-4-ol 834-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidine-4-carbonitrile 844-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-4-ol 854-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 864-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-4-carbonitrile 874-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidine-4-carbonitrile 882-(6-(4-amino-1-(2-hydroxy-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yloxy)acetonitrile 89 methyl2-(6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-2-yloxy)acetate 903-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidin-3-ol 913-((4-amino-3-(6-cyclopropoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylazetidin-3-ol 924-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidine-4-carbonitrile 933-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidin-3-ol 943-(6-(difluoromethoxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 953-(6-(difluoromethoxy)naphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 963-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidin-3-ol 973-((4-amino-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylazetidin-3-ol 984-((4-amino-3-(6-(difluoromethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-4-ol 994-((4-amino-3-(6-(difluoromethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidin-4-ol 1004-((4-amino-3-(6-(difluoromethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-4-carbonitrile 1013-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)azetidin-3-ol 1024-((4-amino-3-(6-(difluoromethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylpiperidine-4-carbonitrile 1033-((4-amino-3-(2-cyclopropoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-1-methylazetidin-3-ol 1041-(3-(6-ethoxynaphthalen-2-yl)-1-((1-(methylcarbamoyl)piperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3-methylurea 1054-((4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-methylpiperidine-1-carboxamide 1063-(2-ethoxyquinolin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine1073-(6-ethoxynaphthalen-2-yl)-1-(2-(piperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1083-(6-ethoxynaphthalen-2-yl)-1-(2-(1-methylpiperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1093-(6-ethoxynaphthalen-2-yl)-1-(2-(1-ethylpiperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1103-(6-ethoxynaphthalen-2-yl)-1-(2-(1-propylpiperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1111-(2-(1-(3-aminopropyl)piperidin-4-yl)ethyl)-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1123-(6-ethoxynaphthalen-2-yl)-1-(1-propylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1131-(1-(3-aminopropyl)piperidin-4-yl)-3-(6-ethoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1141-(6-ethoxynaphthalen-2-yl)-3-(piperidin-4-ylmethyl)imidazo[1,5-a]pyrazin-8-amine 1151-(6-ethoxynaphthalen-2-yl)-3-((1-methylpiperidin-4-yl)methyl)imidazo[1,5-a]pyrazin-8-amine 1161-isopropyl-3-(6-(oxetan-3-yloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 1171-((6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yl)oxy)-2-methylpropan-2-ol 1182-((6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-yl)oxy)ethan-1-ol 1193-(2-ethoxyquinolin-6-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.

Biological Example 1: TgCDPK1 Enzymatic Inhibition Assay

Inhibitors were evaluated in triplicate in eight-point dilutions (3-folddilutions) during the enzymatic reactions. TgCDPK1 enzymatic inhibitionwas determined with a coupled luciferase assay (Kinaseglo®). 2.1 nMTgCDPK1 and 20 μM BioSyntide-2 (American Peptide Company, Inc.Sunnyvale, Calif.)) were incubated in 25 μL of buffer containing 1 mMEGTA (pH 7.2), 10 mM MgCl₂, 20 mM HEPES, pH 7.5 (KOH), 0.1% BSA, and 2mM CaCl₂. The reaction was initiated with the addition of ATP at a 10 μMfinal concentration. After incubating at 30° C. for 90 min., changes inATP concentration were determined by adding Kinaseglo® luciferasereagent (Promega, Madison, Wis.) and measuring luminescence with aMicroBeta2 multi-label plate reader (Perkin Elmer, Waltham, Mass.).Results were converted to percent inhibition, and IC₅₀ values werecalculated using nonlinear regression analysis in GraphPad Prism.

Biological Example 2: Src Kinase Enzymatic Inhibition Assay

Inhibitors were evaluated in triplicate in eight-point dilutions (3-folddilutions) during the enzymatic reactions. Chicken Src enzymaticinhibition was determined with a coupled luciferase assay (Kinaseglo®).2 nM Src and 61 μM Src substrate peptide (GenScript, Piscataway, N.J.)were incubated in 25 μL of buffer containing 40 mM Tris-HCl (pH 7.5), 20mM MgCl₂, 1 mM MnCl₂, 1 mM DTT, and 0.1% BSA. The reaction was initiatedwith the addition of ATP at a 10 μM final concentration. Afterincubating at 30° C. for 90 min., changes in ATP concentration weredetermined by adding Kinaseglo® luciferase reagent (Promega, Madison,Wis.) and measuring luminescence with a MicroBeta2 multi-label platereader (Perkin Elmer, Waltham, Mass.). Results were converted to percentinhibition, and IC50 values were calculated using nonlinear regressionanalysis in GraphPad Prism.

Biological Example 3: Human Cell Growth Inhibition Assay

CRL-8155 human lymphocytic cells (ATCC, WIL2-NS) were cultured inRPMI-1640 growth medium supplemented with 10% heat inactivated fetalbovine serum, 10 mM HEPES, 1 mM sodium pyruvate, and 1 mM L-glutamine.The Alamar Blue® assay (Invitrogen, Grand Island, N.Y.), which measuresgeneral cellular metabolism, was used to quantify cell growth. Mid-logcells were seeded in 96-well flat-bottom plates (Corning, Corning, N.Y.)at a density of 3×10⁵ cells/mL containing test compounds at six finalconcentrations (40 μL, 20 μL, 10 μL, 5 μL, 2.5 μL, and 1.25 μL) inquadruplicate and grown at 37° C. for 48 hours in a 5% CO₂ humidifiedincubator. A 1/10th volume of Alamar Blue® developing reagent was addedto each well and incubated for an additional 3 hours and fluorescencewas measured at the respective excitation and emission wavelengths of560 nm and 590 nm in a FLx800 microplate reader (Biotek, Winooski, Vt.).Percent growth inhibition by test compounds was calculated based on DMSOvehicle and positive controls (50 μL quinacrine), which corresponded to0% and 100% growth inhibition, respectively.

Biological Example 4: T. Gondii Growth Inhibition Assay

The T. gondii growth inhibition assay was performed according to apreviously reported procedure. Briefly, a dilution series of aninhibitor (diluted in DMSO) was added to DMEM (final DMSO=0.5%). T.gondii expressing a β-galactosidase reporter were added to the DMEM andincubated briefly before adding them to fibroblast monolayers in 96 wellplates. Plates were visually inspected for evidence of cytotoxic effectson fibroblasts. After 44 h, β-galactosidase was assayed usingchlorophenol red β-galactopyranose (Sigma-Aldrich, St. Louis, Mo.) as asubstrate. Each dilution series of inhibitor was tested at least twicein triplicate. For assays with drug resistant T. gondii, the sameprocedure was followed except cell lines expressing HA-TgCDPK1 orHA-Gly128Met TgCDPK1 in a “wild type” background were tested.

Biological Example 5: In Vivo Efficacy Against Acute T. Gondii Infectionin Mice

Infection and drug administration were performed as previously reported.Mice were infected with type 1 RH strain T. gondii expressing a yellowfluorescent protein. T. gondii were harvested from human foreskinfibroblasts, passed through a 3-μm-pore filter, and 10 tachyzoites wereinoculated in a volume of 100 μl of phosphate-buffered saline (PBS)intraperitoneally (i.p.) into 4- to 5-week-old, 25-g female CF-1 mice.The compounds were dissolved in polyethylene glycol (PEG) 400 andadministered by oral gavage 48 h after inoculation. The control groupreceived PEG 400 only. Groups consisted of 4 mice. After mice wereeuthanized on the eighth day, the brain and spleen were collected fromthe mice and peritoneal lavage was performed with 3 ml of PBS (pH 7.4).

In vivo efficacy was evaluated with quantitative real-time PCR for T.gondii DNA from the brain and spleen, and quantification of peritonealT. gondii infection as previously described. A sample of 10 μl ofperitoneal lavage fluid was examined in a hemocytometer usingfluorescence microscopy (excitation/emission 480/535 nm).Yellow-fluorescent tachyzoites were quantified per mL of fluid. Afterthe mice were euthanized, the entire brain and spleen were collected andhomogenized. DNA was isolated with a DNA purification kit (Qiagen,Germantown, Md.). 300 ng of total DNA from the brain homogenate and 200ng of total DNA from the spleen homogenate were analyzed per mouse. Astandard curve was generated from DNA purified from T. gondiitachyzoites in 10-fold dilutions from 160 ng to 1.6 fg of DNA.Quantitative real-time PCR was performed in duplicate using a 7300Real-Time PCR System (Applied Biosystems, Grand Island, N.Y.) with iTaqSYBR GREEN PCR Supermix and primers for the T. gondii 529-bp repeatelement. Results were quantified as T. gondii DNA per total DNA.Analysis was performed with GraphPad Prism 5.0 software. This protocolwas approved by the institutional animal care and use committee of thePortland Veterans Administration Medical Center.

Biological Example 6: Pharmacokinetic Analysis in Mice and Rats

For mouse oral PK studies, three female BALB/c mice (10 to 12 weeks old)were used in each group. Each group received a test compound at a doseof 10 mg/kg body weight dissolved in 3% ethanol/7% Tween 80/90% normalsaline by oral gavage. Blood samples were taken at the designated timepoints by tail bleeding and centrifuged to obtain plasma. The sampleswere frozen at −20° C. The test compounds were extracted from the plasmasamples using acetonitrile/0.1% formic acid with an internal standard. Astandard mix of all test compounds was prepared for comparison andquantification. The compounds were quantified by LC/MS analysis. PKcalculations were performed using Phoenix WinNonlin software(Pharsight).

In rats, test compound was administered to Sprague-Dawley jugularcanulated rats (Charles River) by either oral gavage or IV injectionfollowed by blood sampling from the jugular vein at designated timepoints. The oral dose was administered to each rat at 20 mg/kg forcompound Ex. 24 and 5 mg/kg for compound Ex. 25 at time=0 in a 1 mLvolume of dosing solution (7% Tween 80, 3% EtOH, 5% DMSO, 0.9% saline.)IV injections were administered at 5 mg/kg from time=0 to 3 minutes in a1 mL volume of dosing solution, and blood was sampled at the same timepoints via the jugular vein. Experiments were performed with groups of 2rats each for the oral and IV dosing. Plasma was separated and extractedwith acetonitrile and quantified by LC/MS analysis. PK calculations wereperformed using Phoenix WinNonlin software (Pharsight).

Biological Example 7: Distribution of Compounds Between Mouse Plasma andBrain

Mice were injected with test compounds (5 mg/kg IP) and sacrificed atthe indicated times for collection of plasma and brain. Compound wasdissolved in 0.4 mL of dosing solution (7% Tween 80, 3% ethanol, 5%DMSO, 0.9% saline) for IP injections. The brains were weighed andimmediately frozen, then later homogenized in acetonitrile. Prior toanimal studies, recovery of test compound was carried out by adding aknown amount to a mouse brain in the test extraction solvent andperforming the homogenization. Compound recovery was determined byliquid chromatography/tandem mass spectrometry analysis relative to astandard compound amount. Blood was taken from the same mice inheparinized capillary tubes for determination of compound concentrationin plasma. The concentration of compound in the brain was obtained bydividing the moles of compound in the brain by the brain volume(obtained from the brain weight assuming 1 g is 1 mL) and correcting forthe brain vasculature volume of 3% by weight.

Results

The following compounds were tested for inhibition TgCDPK1:

TABLE 1 Ex. No. TgCDPK1 IC50 (μM) 1 0.0025 2 0.0172 3 0.0016 4 0.0015 50.0045 6 0.0056 7 0.0021 8 0.0949 9 0.0060 10 0.0040 11 0.0155 12 0.010713 0.0463 14 0.0242 15 0.0061 16 0.0022 17 0.0008 18 0.0012 19 0.0013 200.0018 21 0.0024 22 0.0018 23 0.0033 24 0.0010 25 0.0020 26 0.0009 270.0212 29 0.0048 30 0.0026 31 0.0358 32 0.0012 33 0.0057 34 0.0058 350.0040 36 0.0017 37 0.0017 38 0.0038 39 0.0010 40 0.0023 41 0.0028 420.0080 43 0.0020 44 0.0014 45 1.1991 46 0.0183 47 0.0108 48 0.0191 490.0029 50 0.0429 51 0.0158 52 0.0842 53 0.0473 54 1.5500 55 0.0032 560.0708 57 0.0061 58 0.0091 59 0.0086 60 0.0127 61 0.0130 62 0.0192 630.0128 64 0.0036 65 0.0029 66 1.6629 67 0.4295 68 0.0097 69 0.0432 700.0233 71 0.0593 72 0.0496 73 0.0738 74 0.0027 75 0.0183 76 0.0194 770.0064 78 0.0007 79 0.0045 80 0.0231 81 0.0013 82 0.0007 83 0.0035 840.0040 85 0.0210 86 0.0015 87 0.1030 88 0.1124 89 1.4400 90 0.0051 910.0038 92 0.0055 93 0.0029 94 0.0019 95 0.0065 96 0.0078 97 0.0026 980.0075 99 0.0358 100 0.0025 101 0.0030 102 0.0182 103 0.0048 106 0.0020107 0.0019 108 0.0022 109 0.0027 110 0.0030 111 0.0043 112 0.0043 1130.0043 114 0.0100 115 0.0186 116 0.0061 117 0.0030 118 0.0062 119 0.0034

Based on the abilities of a number of inhibitors to potently block T.gondii proliferation in mammalian cells, while demonstrating little orno off-target toxicity or hERG inhibition, the solubility in water andpharmacokinetic (PK) properties were examined (Table 2). The solubilityof selected inhibitors was determined at pH=6.5. The initial PK profilesof these inhibitors were determined after a single 10 mg/kg oral dose inthree Balb/c mice, with sampling conducted at the time points indicatedin the Experimental Section. As a reference, after a 10 mg/kg po dose,previously reported inhibitor3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(identified as compound 150 in US 2013/0018040, and hereafter) has amaximum concentration (Cmax) of 0.75±0.15 μM and total exposure (areaunder the plasma concentrations versus time curve, AUC) of 430±84μM·min. For example Compounds of example 23-25, which all contain a2-methylpropan-2-ol at the R2 position, are highly soluble and 24 and 25reach maximum serum concentrations (Cmax)>10-fold higher and totalexposure >30-fold higher than1-(4-amino-3-(2-ethoxyquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-2-olpreviously reported compound 150 (Table 2 and FIG. 1).

TABLE 2 Solubility and PK properties of potent and selective TgCDPK1inhibitors dosed at 10 mg/kg PO to mice. Solubility Cmax AUCo->inf Halflife clearance Ex. No. (μM) Tmax (min) (μM) (μM · min) (min) (mL/min) 2217 30 0.88 ± 0.23  67 ± 60  50 ± 73 110 23 >100 50 5.2 ± 1.0  850 ± 411 114 ± 115 0.2 24 >100 320 13 ± 1  13700 ± 1500 1190 ± 510 0.2 25 >100560 7.8 ± 1.4 16600 ± 4300 1110 ± 400 0.3 26 6.2 40 0.39 ± 0.16 31 ± 840 ± 5 6.7 27 8.0 30 3.9 ± 1.1  3.9 ± 1.1  41 ± 21 21

Based on the PK parameters of 24 and 25 following single doseadministration to mice, these compounds were evaluated for tolerabilityfollowing escalated dosing. Both compounds were initially dosed at 10mg/kg on day one. As no observable adverse effects were detected in micedosed with either 24 or 25, the next dose was increased to 50 mg/kg onday 4, followed by a final dose of 100 mg/kg on day 8. Mice showed noovert signs of toxicity or weight loss over the 10-day observationperiod. Next, the plasma protein binding of both compounds wasevaluated. Both compounds were highly protein bound in mouse plasma withplasma protein binding of 96% for 24 and 88% for 25. Based on theprotein binding values the plasma concentrations required to maintainunbound plasma exposure above the TgCDPK1 EC50 are 1.33 μM for 24 and1.5 μM for 25. Based on these target concentrations and the single dosePK data, a multiple dosing regimen with a loading dose of 20 mg/kg of 24and 25 followed by a 5 mg/kg daily dose for five days was evaluated.Blood was collected at multiple time points to determine plasmaconcentrations over the course of treatment (FIG. 2). Both compoundsdemonstrated excellent exposure through the 24-hour dosing interval withthe trough concentrations remaining >1.5 μM throughout the study. Themeasured concentrations following multiple dosing were similar to thosepredicted from the single dose PK studies in mice (FIG. 3). Neithercompound showed any evident signs of toxicity compared to mice dosedwith vehicle only. Cardiac puncture blood collection was performed atthe end of the study for complete blood count and serum biochemicalprofiles. All results were reported by Phoenix Central Laboratory to bewithin a normal range for species and age.

In a second multiple dose study compound 24 was administered at 50 mg/kgpo every other day for 5 doses and compound 25 was administered with a20 mg/kg loading dose followed by a 10 mg/kg maintenance dose everyother day for 5 doses. The exposure to 25 with this dosing regimen wassimilar to the one observed with daily dosing of the lower dose and theplasma concentration at various time points were well predicted based onthe single dose PK results. With compound 24 plasma concentrationsremained >10 μM for the entire duration of the study (FIG. 2) withmaximum concentrations reaching 43 μM. The exposure to compound 24following the last 50 mg/kg dose was higher than predicted from singledose studies with longer half-life suggesting possible saturation ofmetabolism at this dose.

Based on the favorable single and multiple dose PK and the lack ofobservable toxicity, compounds 24 and 25 were evaluated for tolerabilityand PK characteristics in rats following intravenous (IV) and PO dosing.Following IV administration to rats both 24 and 25 displayed biphasickinetics with compound 25 reaching distribution equilibrium more rapidlythan 24. Compound 24 distributed approximately to total body water witha volume of distribution at steady state (Vss) of 0.9 L/kg. Thedistribution of 25 was more extensive with a Vss of 5.8 L/kg. Bothcompounds had very low systemic clearances in rats but, in contrast tomice, the clearance of 25 was 4-fold higher than that of 24. Theelimination half-lives of both compounds were acceptable for multipledosing regimens, 9.6 and 13 hours for 24 and 25, respectively (FIG. 3).

Following oral administration to rats both compounds showed slowabsorption with absorption phase continuing for 12 hours after oraldosing. The plasma concentrations over the 24-hour period after POdosing exceeded those observed after IV dosing (FIG. 3) suggesting thatboth 24 and 33 had essentially complete bioavailability in rats. Theapparent bioavailability of these compounds was greatly improvedcompared to that of compound 150 (46%)

Because it is important that anti-toxoplasmosis therapies are able toprevent reactivation of parasites within tissue cysts, which largelyreside in the central nervous system (CNS), the distribution ofcompounds 24 and 25 to the brain was next determined. To do this, thedistribution of 24 and 25 into mouse brain (n=3) at one hr afterintraperitoneal dosing of 5 mg/kg was measured. The mean concentrationof compound 24 at one hour in brain was 1.2±0.5 μM, a concentration wellabove the TgCDPK1 EC₅₀. The corresponding plasma concentration of 24 was4.1±1.1 μM resulting in a brain to plasma concentration ratio of 24 of0.33±0.22. This brain penetration was comparable to compound 150 (0.31).In accordance with the larger distribution volume of compound 25, itdemonstrated a greater brain to plasma concentration ratio (1.65±0.84).Both the brain and plasma concentration of 25, 1.90±0.24 μM and1.23±0.76 μM, respectively, were above the TgCDPK1 EC₅₀.

To demonstrate that TgCDPK1 is the kinase target of these compounds inT. gondii, 24 and 25 were tested against parasitic cell linesoverexpressing the Gly128Met TgCDPK1 gatekeeper mutant or wildtype (wt)TgCDPK1. Expression of the Gly128Met gatekeeper mutant of TgCDPK1, butnot wtTgCDPK1, makes T. gondii highly resistant to PP-based inhibitorsthat contain 6-alkoxynaphthalen-2-yl groups at the R₃ position. Both 24and 25 show a dramatic loss in potency against parasites overexpressingthe Gly128Met gatekeeper mutant relative to the parent RH strain and toT. gondii overexpressing wild type TgCDPK1 (FIG. 4), which is consistentwith TgCDPK1 being the primary target through which these inhibitorsexert their anti-parasitic effects. 24 and 25 were further profiled forany mammalian kinase off targets using a panel of 80 human kinasesrepresenting different subfamilies of the kinome tree with afluorescence-based competition assay. 78 of the 80 mammalian kinasestested have an IC₅₀>1.5 μM (>1500-fold selective for TgCDPK1) for 24.For the two kinasesPKCv (PKD3) and MEK1 that have sub-micromolar IC50values, compound 24 is a >120-fold and >900-fold less potent inhibitorthan for TgCDPK1, respectively. Compound 25 appears to be slightly moreselective than compound 24. Compound 25 demonstrated an IC50 value ofgreater than 5 μM (>2500-fold selective) for 79 of the kinases tested,with only PKCv (PKD3) demonstrating a submicromolar (IC₅₀=0.280 μM;140-fold selective) IC₅₀ value.

The lack of toxicity in the initial mouse toxicity screens allowed us tomove forward into large animal pharmacokinetic and tolerabilityprofiling. Male calves (n=2 for each compound) were dosed orally at 10mg/kg for compound 24 and 9.3 mg/kg for compound 25 and blood sampled upto 12 days after dosing (results not shown). Similar to rats, theabsorption of both compounds was slow with maximum concentrationsreached at 24 hours for compound 24 and 12 hours for compound 25. Themaximum plasma concentrations were similar for the two compounds, 7.9 μMand 9.8 μM, for compound 24 and 25, respectively. However, the overallexposure to 25 was greater than that of 24 due to its lower oralclearance and longer half-life (Table 3). Yet, both compounds had verylow oral clearances and long systemic half-lives. Similar to rats,compound 25 had a higher apparent volume of distribution than compound24 and the overall distribution characteristics were similar to thoseobserved in rats.

TABLE 3 In vivo pharmacokinetic parameters of 24 and 25 in calvesfollowing PO administration of 10 mg/kg of 24 and 9.3 mg/kg of 25. Dataare shown as the mean and range between animals. AUC CL/F V/F Ex.(h*μmol/L) (mL/hr/kg) (L/kg) t½ (hr) 24 588 (353-825)  52 (31-72) 1.5(1.3-1.8) 23 (17-28) 25 849 (607-1091) 30 (22-39) 2.3 (1.4-3.2) 51(46-56)

Potential further toxicity of compound 24 was examined in mice bytesting two doses (30 mg/kg and 100 mg/kg PO) daily for five days, whileobserving mice for signs of toxicity and collecting blood samples. Bothgroups of mice remained active, well groomed, and appeared normalthroughout the study. Upon necropsy, there were no gross abnormalities.Histology revealed mild focal inflammation in the spleen in two of threemice in the 30 mg/kg group. The only abnormality seen in the 100 mg/kggroup was inflammatory infiltrate in the hepatic lobules of the liver inone of three mice. The concentrations following 30 mg/kg doses wereslightly higher than those predicted from single dose PK data while theexposures following the 100 mg/kg doses were similar to those predictedfrom single 10 mg/kg dose data (FIG. 5). Similar to the early multipledosing experiments in mice, after the last 100 mg/kg dose theelimination of compound 24 was slower than predicted with considerableplasma concentrations persisting at 72 hours after the final dose.

Mice were then dosed with single PO doses of 24 ranging from 200 mg/kgto 1000 mg/kg. The lowest observable adverse effect level (LOAEL) wasobserved at 500 mg/kg and the no observable adverse effect level (NOAEL)was observed at 400 mg/kg. Mice had slightly ruffled fur and were lessactive than the control mice at 3 hours following the 500 mg/kg dose.The lowered activity persisted at 24 hours but was resolved by 30 hours.The lack of toxicity up to 500 mg/kg confirmed the safety of thecompound for small animal efficacy studies.

The pharmacokinetics of 24 was further explored in dogs and monkeysfollowing IV and PO administration. Similar to rats, 24 had a very lowclearance in both species and a relatively long half-life (Table 4). Theplasma concentrations time-profile following IV dosing was biphasic inboth species with a similar extent of distribution in dogs and monkeysas observed in rats and calves (FIG. 3, panel B). The bioavailability ofcompound 24 was 66±17% in monkeys and 88±12% in dogs demonstrating goodbioavailability in both species as predicted from rats.

TABLE 4 In vivo pharmacokinetic parameters of 24 following IV and POadministration of 1 mg/kg to dogs and monkeys (n = 3 for each). Data isshow as mean and standard deviation. AUC (h*μ mol/L) CL (mL/hr/kg) Vss(L/kg) t½ (hr) Dog IV 11200 ± 1100  90 ± 10 1.7 ± 0.1 13.2 PO 9620 ±1350 Monkey IV 8720 ± 3400 130 ± 50  1.8 ± 0.3 9.6 PO 5730 ± 1500

Example compound 24 was tested against a high inoculum of type 1 RHstrain T. gondii to determine efficacy against fulminate toxoplasmosisin two experiments (FIG. 6). Unlike type 2 T. gondii strains, type 1strains do not typically form tissue cysts but rather cause death in5-10 days depending on the size of the inoculum. Compound 24 wasadministered via oral gavage two days after infection and the burden ofinfection was measured 5 days after the initiation of treatment.Treatment with 24 at 20 mg/kg for 5 days reduced the T. gondii in theperitoneal fluid below the limits of detection (less than 100tachyzoites/mL). Treatment with 24 was highly effective at 20 mg/kgdaily for 5 days in reducing infection in the spleen more than 99%, andinfection in the brain 95%. The marked decrease in brain infectioncompared to controls coupled with the measurement of a sufficient brainconcentrations of 24 suggest that this compound is active againstactively replicating T. gondii in the brain, the target of currentfirst-line anti-toxoplasma drugs. Previous mouse models of RH strain T.gondii in mice have detected T. gondii DNA in the brain after 2 days andrecovery of T. gondii in culture at 4 days. In this study, decreases inbrain infection may in part be due to efficacy against systemicinfection. The favorable safety profile and brain permeability of 24make it an attractive candidate to treat toxoplasmosis in pregnancy, aswell as CNS toxoplasmosis.

Biological Example 8: Sarcocystis Neurona Calcium-Dependent ProteinKinase 1 is Targeted in Selective Therapeutic Development for EquineProtozoal Myeloencephalitis

Equine protozoal myeloencephalitis (EMP) is a common progressive,degenerative neurological disease of the central nervous system causedby the apicomplexan parasite Sarcocystis neurona. Widespread therapeuticfailure or relapses even with long term use of available treatments andabsence of viable vaccines underscore the need to validate moleculartargets within the parasite for new drug development based on novelscaffolds with desirable therapeutic outcome. We recently identified andsequenced an equivalent CDPK homologue (SnCDPK1) in S. neurona genomethat has similar glycine “gatekeeper” residue. SnCDPK1 and TgCDPK1have >85% amino acid identity. We have characterized BKIs for in vitroefficacy against SnCDPK1 and S. neurona merozoites. Recombinant SnCDPK1was expressed, purified and screened against a selected group of BKIspreviously shown to have low IC₅₀s against TgCDPK1 and T. gondiitachyzoites. Growth assays with a yellow fluorescent protein-expressingclone of S. neurona demonstrated that parasite growth was inhibited byBKIs at nanomolar concentrations (Table 4).

BKI-CDPK1 binding confirmation was performed using S. neurona lysates inthermal shift assays using CDPK1-specific antibody. SnCDPK1 wasinhibited by low nM concentrations of BKIs. Analysis of Sarcocystiscell-inhibition data suggests that BKI interfered with an early step inS. neurona host cell invasion and egress processes. This presentsmolecular and phenotypic evidences that SnCDPK1 could be targeted forrational drug development as TgCDPK1 was previously validated for T.gondii. BKIs used in these assays have been chemically optimized withfunctional groups needed to improve potency, selectivity andpharmacokinetic properties. In vivo experiments were performed in murinemodel of infections using Sarcocystis neurona strain SN 37R.Experimental group treated with compound of Example 24 at a dose of 10mg/kg/day/oral in drinking water having 0.5% Saccharine for 30 daysshowed no sign of disease relative to the control group 40 days afterthe end of treatment period.

TABLE 4 In vitro activities against SnCDPK1. Thermal SnCDPK1 S. neuronashift from Example No. IC₅₀(μM) STDEV^(n=x) EC₅₀ (μM) DMSO  24   0.00890.0015^(n=4) 0.042 4.1  25   0.0075 0.0007^(n=2) 0.128 ND

>2 0.3557^(n=3) >10 0 150   0.0059 0.0005^(n=3) 0.068 2.7

Biological Example 9: Treatment of Non-Pregnant and Pregnant 248 Balb/cMice Experimentally Infected with the N. Caninum Isolate

The materials and methods for assessing treatment of N. caninum invitro, and treats infected mice and rids of N. caninum infection inbrain and fetuses has been previously described in Winzer et al. “Invitro and in vivo effects of the bumped kinase inhibitor 1294 in therelated cyst-forming apicomplexans Toxoplasma gondii and Neosporacaninum.” Antimicrobial Agents and Chemotherapy 59(10):6361-74 (October2015), incorporated herein by reference in its entirety.

Compound 150 is effective in treating N. caninum in vitro, and treatsinfected mice and rids of N. caninum infection in brain and fetuses. Inaddition, compound 24 has similar properties in that it is active invitro against N. caninum cultures, and it treats pregnant mice removingN. caninum from their brains, their bodies, and their fetuses. A varietyof other compounds have been found to be active against the presumedtarget, N. caninum calcium-dependent protein kinase 1 (cloned andexpressed by our group and was also found to have a glycine gatekeeperresidue), the compounds that are active against T. gondii CDPK1 arealmost all uniformly active against NcCDPK1, and are presumed active inthe in vitro and in vivo models as well.

Biological Example 10: Besnoitia Besnoiti Activity

Besnoitiosis is a chronic and debilitating bovine disease caused by theapicomplexan Besnoitia besnoiti, a protozoan parasite belonging to thegroup of cyst-forming coccidians. There are currently no therapeuticremedies for this disease. Apicomplexan calcium dependent proteinkinases, necessary for host cell invasion and egress, are promisingtargets for drug development because orthologs are absent in mammaliangenomes. Unlike the mammalian host cell kinases, BbCDPK1 has glycine atthe entry to the ATP binding site (gatekeeper residue), which renders itsensitive to bumped kinase inhibitors (BKIs). The activity of two BKIsof the disclosure against Besnoitia besnoiti is provided in Table 5.

TABLE 5 In vitro activities against BbCDPK1 and Besnoitia besnoiti.BbCDPK1 IC₅₀ B. besnoiti EC₅₀ B. besnoiti EC₁₀₀ Example (μM) (μM) (μM)150 0.004 0.045 2.580 24 0.005 0.097 3.590

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

What is claimed:
 1. A compound of formula:

or a pharmaceutically acceptable salt thereof, wherein X, Y, and Z aredefined by either: (i) X is N, Y is C, and Z is N; or (ii) X is C, Y isN, and Z is C(H); R¹ is C₂₋₆ alkyl, C₁₋₆ haloalkyl, —C₁₋₆ alkyl-R¹²,C₃₋₈ cycloalkyl, heterocyclyl, heteroaryl, or aryl, wherein the alkyl,cycloalkyl, heterocyclyl, heteroaryl, and aryl groups are eachoptionally substituted with one or two R¹¹ groups; each R¹¹ isindependently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR,—SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂NR₂, or —S(O)₂R; and R¹² is—OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR,—OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, —N(R)C(O)NR₂, aryl, heteroaryl, C₃₋₈cycloalkyl, or heterocyclyl, wherein R¹² is optionally substituted byone, two, or three groups that are each independently halogen, cyano,nitro, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ hydoxyalkyl, —OR, —SR, —NR₂,—C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,—N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂; R³ is one of the formulas,

wherein n is 0, 1, or 2; Q is —O—, —S—, or —N(R^(Q))—, wherein R^(Q) ishydrogen or C₁₋₆ alkyl; and R³³ is C₁₋₆ alkyl or (C₃₋₈ cycloalkyl)C₁₋₆alkyl, wherein the alkyl and cycloalkyl are optionally substituted withone, two, three, or four groups that are each independently halogen,cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂,—C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,—OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂,wherein each R²⁰ is independently hydrogen or C₁₋₆ alkyl, each R³² isindependently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR³⁴,—SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴,—OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴,or —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is independently hydrogen orC₁₋₆ alkyl; and each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆alkyl, heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl,arylalkyl, heteroaryl, and heteroarylalkyl are optionally substitutedwith one, two, three, or four groups that are each independentlyhalogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR⁰, —SR⁰, —N(R⁰)₂,—C(O)R⁰, —C(O)OR⁰, —C(O)N(R⁰)₂, —S(O)₂R⁰, —OC(O)R⁰, —OC(O)OR⁰,—OC(O)N(R⁰)₂, —N(R⁰)C(O)R⁰, —N(R⁰)C(O)OR⁰, or —N(R⁰)C(O)N(R⁰)₂, whereineach R⁰ is independently hydrogen or C₁₋₆ alkyl; provided the compoundis not:1-(6-ethoxynaphthalen-2-yl)-3-isopropylimidazo[1,5-a]pyrazin-8-amine;3-(6-isopropoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-ethoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-methoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-isopropoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-(piperidin-4-ylmethyl)-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(benzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-butoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(allyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(allyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-butoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-isobutoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-isobutoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2,5-dimethylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(2-methylbenzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(2-methyl-5-(trifluoromethyl)benzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chloro-4-(2,2,2-trifluoroethyl)benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chloro-5-fluorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(1-phenylethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-tert-butylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(pyridin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N,N-dimethylquinolin-2-amine;3-tert-butyl-1-(6-ethoxynaphthalen-2-yl)imidazo[1,5-a]pyrazin-8-amine;3-tert-butyl-1-(6-methoxynaphthalen-2-yl)imidazo[1,5-a]pyrazin-8-amine;3-(6-ethoxynaphthalen-2-yl)-1-(1-ethylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;or3-(6-ethoxynaphthalen-2-yl)-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.2. The compound of claim 1, which is of the formula


3. The compound of claim 1, wherein R³ is of the formula


4. The compound of claim 1, wherein Q is —O—.
 5. The compound of claim1, wherein R¹ is C₂₋₆ alkyl or —C₁₋₄ alkyl-R¹².
 6. The compound of claim5, wherein R¹² is —OR or heterocyclyl, each optionally substituted.
 7. Apharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable excipient, carrier, or diluent.
 8. A methodof treating an apicomplexan protozoan related disease, wherein theapicomplexan protozoan related disease is selected from toxoplasmosis,cryptosporidiosis, coccidiosis, malaria, neosporosis, sarcocystosis,besnoitiosis, coccidiosis, cystoisoporosis, babesiosis, andtheileriosis, comprising administering to a patient in need of suchtreatment a therapeutically effective amount of (i) a compound of claim1, or (ii) a pharmaceutical composition comprising such compound and apharmaceutically acceptable excipient, carrier, or diluent.
 9. Themethod of claim 8, wherein the apicomplexan protozoan related disease isselected from toxoplasmosis, cryptosporidiosis, coccidiosis, andmalaria.
 10. The method of claim 8, wherein the apicomplexan protozoanrelated disease is selected from toxoplasmosis or cryptosporidiosis. 11.A method of treating malaria comprising administering to a patient inneed of such treatment a therapeutically effective amount of (i) acompound of claim 1 or (ii) a pharmaceutical composition comprising suchcompound and a pharmaceutically acceptable excipient, carrier, ordiluent.
 12. A method of treating an apicomplexan protozoan relateddisease, wherein the apicomplexan protozoan related disease is selectedfrom toxoplasmosis, cryptosporidiosis, coccidiosis, malaria,neosporosis, sarcocystosis, besnoitiosis, coccidiosis, cystoisoporosis,babesiosis, and theileriosis, comprising administering to a patient inneed of such treatment a therapeutically effective amount of apharmaceutical composition comprising: (i) a compound selected from3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,or3-(2-ethoxyquinolin-6-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;and (ii) a pharmaceutically acceptable excipient, carrier, or diluentselected from: calcium carbonate, sodium carbonate, lactose, calciumphosphate, sodium phosphate, starch or corn starch, alginic acid,gelatin, acacia, magnesium stearate, stearic acid, talc, glycerylmonostearate, glyceryl distearate, gelatin, kaolin, an oil medium,sodium carboxymethylcellulose, methylcellulose,hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth, gum acacia, lecithin, polyoxyethylene stearate,heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate,polyethylene sorbitan monooleate, glycerol, propylene glycol, sorbitol,glucose, sucrose, and combinations thereof, and optionally one or morepreservative.
 13. The method of claim 11, for treating neosporosis,sarcocystosis, besnoitiosis, cystoisoporosis, or theileriosis.
 14. Themethod of claim 11, wherein the compound is3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.15. The method of claim 11, wherein the compound is3-(2-ethoxyquinolin-6-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.16. A method of treating an apicomplexan protozoan related disease,wherein the apicomplexan protozoan related disease is selected fromtoxoplasmosis, cryptosporidiosis, coccidiosis, malaria, neosporosis,sarcocystosis, besnoitiosis, coccidiosis, cystoisoporosis, babesiosis,and theileriosis, comprising administering to a patient in need of suchtreatment one or more dosage units of a pharmaceutical compositioncomprising: (i) a compound selected from3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,or3-(2-ethoxyquinolin-6-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,wherein the dosage of the compound in the dosage unit is 1 mg to 100 mgper kg of the patient; and (ii) a pharmaceutically acceptable excipient,carrier, or diluent.
 17. The method of claim 16, for treatingneosporosis, sarcocystosis, besnoitiosis, cystoisoporosis, ortheileriosis.
 18. The method of claim 16, wherein the compound is3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.19. The method of claim 16, wherein the compound is3-(2-ethoxyquinolin-6-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine.20. The method of claim 16, wherein the compound is present in a dosageof 5 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg.